Aza-benzazolium containing cyanine dyes

ABSTRACT

Unsymmetrical cyanine dyes that incorporate an aza-benzazolium ring moiety are described, including cyanine dyes substituted by a cationic side chain, monomeric and dimeric cyanine dyes, chemically reactive cyanine dyes, and conjugates of cyanine dyes. The subject dyes are virtually non-fluorescent when diluted in aqueous solution, but exhibit bright fluorescence when associated with nucleic acid polymers such as DNA or RNA, or when associated with detergent-complexed proteins. A variety of applications are described for detection and quantitation of nucleic acids and detergent-complexed proteins in a variety of samples, including solutions, electrophoretic gels, cells, and microorganisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/557,065, filed Jul. 24, 2012, which is a continuation of U.S.application Ser. No. 12/984,380, filed Jan. 4, 2011 (now U.S. Pat. No.8,252,530), which is a continuation of U.S. application Ser. No.11/756,765, filed Jun. 1, 2007 (now U.S. Pat. No. 7,871,773), which is acontinuation of U.S. application Ser. No. 10/683,753, filed Oct. 13,2003 (now U.S. Pat. No. 7,226,740), which is a division of U.S.application Ser. No. 09/557,275, filed Apr. 24, 2000 (now U.S. Pat. No.6,664,047), which claims priority to U.S. Provisional Application Ser.No. 60/131,782 filed Apr. 30, 1999, and U.S. Provisional ApplicationSer. No. 60/158,859, filed Oct. 12, 1999, which disclosures are hereinincorporated by reference in their entirety.

INTRODUCTION

1. Field of the Invention

The invention relates to cyanine dyes that possess particular utility asfluorescent stains for nucleic acids and poly(amino acids). Inparticular, the invention relates to materials that are unsymmetricalcyanine dyes incorporating an aza-benzazolium moiety. The subject dyes,which form fluorescent complexes in combination with nucleic acids orlipid-complexed poly(amino acids), can be used in analyzing a wide rangeof materials, including biological and environmental samples.

2. Background of the Invention

As researchers in the fields of molecular and cell biology increasinglyutilize fluorescent probes as research tools, the ability to select thewavelength of fluorescence is becoming critical. As increasing numbersof multiple-color applications are developed, the ability to producenovel fluorescent probes with detectably distinct fluorescent signals isbecoming ever more important.

Unsymmetrical cyanine dyes have previously been used as fluorescentstains for nucleic acids, see for example U.S. Pat. No. 4,883,867 toLee, at al. (1989) and U.S. Pat. No. 4,937,198 to Lee, et al. (1990).The general spectral properties of the previously described cyanine dyescould be selected somewhat by changing the number of methine groups inthe dye, and whether the dye incorporated a pyridinium or quinoliniumring system (see, for example, U.S. Pat. No. 5,321,130 to Yue et al.(1994); U.S. Pat. No. 5,410,030 to Yue at al. (1995); U.S. Pat. No.5,436,134 to Haugland at al. (1995); U.S. Pat. No. 5,582,977 to Yue etal. (1996); U.S. Pat. No. 5,658,751 to Yue et al. (1997); and U.S. Pat.No. 5,863,753 to Haugland et al. (1999). In particular, monomethine dyesthat incorporate pyridinium ring systems typically exhibit blue toblue-green fluorescence emission, while those that incorporatequinolinium ring systems exhibit green to yellow-green fluorescenceemission. Trimethine dyes are substantially shifted toward redwavelengths, and pentamethine dyes are typically shifted even further,and may exhibit infrared fluorescence emission.

While the spectral properties of cyanine dyes can be finely adjusted byselection of appropriate dye substituents, there were neverthelessregions of the visible spectrum where suitable fluorescent cyanine dyesthat were useful as nucleic acid stains either did not exist, or did notpossess particularly favorable fluorescence properties.

The dyes of the invention incorporate additional nitrogen atoms in thearomatic benzazolium portion of the dye. The dyes of the inventionexhibit a bathochromic spectral shift (a shift to longer wavelength) ofapproximately 30 to 50 nm relative to otherwise structurally similarcyanine dyes known in the art. This bathochromic spectral shift yieldsdyes that are particularly useful for excitation in the wavelengthranges between 500 nm and 600 nm and at >630 nm. Of particularimportance are the dyes of the invention that exhibit absorbance maximabetween 530 nm and 550 nm, as they match the principal emission lines ofthe mercury arc lamp (546 nm), frequency-doubled Nd-Yag laser (532 nm),and HeNe laser (543 nm).

Styryl dyes that complex with lipid-complexed poly(amino acids) havebeen described previously (U.S. Pat. No. 5,616,502 to Haugland et al.,1997). Cyanine dyes that incorporate a 3,4-diazaindene ring system havebeen described previously for use as optical sensitizers in photographicmaterials (British patent No. 870,753 to Ficken et al., (1961)), buttheir use in association with either nucleic acids or lipid-complexedproteins as fluorescent stains has not previously been described.

SUMMARY OF THE INVENTION

The present invention provides unsymmetrical aza-benzazolium containingcyanine dyes (monomers and dimers), reactive versions of the dyes,dye-conjugates and methods for detecting the presence of an analyte ofinterest in a sample. The analyte of interest is typically a nucleicacid or a poly(amino acid) wherein the present dyes in the form of astaining solution are combined with the sample for a sufficient amountof time to form a dye-analyte complex and are illuminated with anappropriate wavelength whereby the nucleic acid or a poly(amino acid) isdetected.

The dyes of the invention form a staining solution by typically beingdissolved in an organic solvent such as DMSO to form a stock solutionand then diluted to an appropriate concentration in a buffered solution.The staining solution is then incubated with a sample to form thedye-analyte complex.

In one aspect of the invention the analyte is nucleic acids that may bepresent in solution, immobilized on a solid or semi solid matrix orpresent in a biological structure. Typically the nucleic acid isimmobilized on solid or semi solid matrix that is selected from thegroup consisting of a polymeric gel, a membrane, an array, a glass slideand a polymeric particle. Preferably the nucleic acid is immobilized ona glass slide or in a gel such as an agarose gel. Alternatively, thenucleic acid is present in a biological structure that is a biologicalcell or portion thereof, a virus particle or a tissue section.

In another aspect of the invention the analyte is a poly(amino acid),typically the poly(amino acids) are complexed with a lipid, such as ananionic detergent. Therefore, the poly(amino acid) is typically presentin solution or immobilized on or in a solid- or semi-solid matrixwherein the matrix is a polymeric gel, a membrane, an array or apolymeric particle. Typically the poly(amino acid) is immobilized on apolyacrylamide gel wherein the staining solution is combined before,during or after immobilization on the gel. Preferably the poly(aminoacid) are electrophoretically separated on the polyacrylamide gel.Alternatively, the poly(amino acids) may be spotted onto apolyacrylamide gel surface such as a Hydrogel microarray slide.

Typically, the dyes of the invention are according to the followingformula;

-   -   wherein A represents the atoms necessary to form one to two        fused aromatic rings having 6 atoms in each ring, at least one        of which is a nitrogen atom, said ring or rings being optionally        further substituted one or more times by C₁-C₆ alkyl, C₁-C₆        alkoxy trifluoromethyl, halogen, BRIDGE, -L-Rx or -L-Sc;        -   wherein, Rx is a reactive group; Sc is a conjugated            substance; and L and BRIDGE are independently a single            covalent bond, or a covalent linkage;    -   X is O, S, Se, NR¹⁵, or CR¹⁶R¹⁷, where R¹⁵ is H or an alkyl        group having 1-6 carbons; and R¹⁶ and R¹⁷, which may be the same        or different, are independently alkyl groups having 1-6 carbons,        or R¹⁶ and R¹⁷ taken in combination complete a five or six        membered saturated ring;    -   R² is selected from the group consisting of -L-Rx, -L-Sc, TAIL,        BRIDGE and an alkyl group having 1-6 carbons that is optionally        substituted by sulfa, carboxy, amino, substituted amino or        substituted ammonium, wherein α is 0 or 1; and TAIL is a        heteroatom-containing moiety;    -   Y is -CR³=CR⁴- wherein p and m=0 or 1, such that p+m=1;    -   R³, R⁴, R⁶, and R⁷ are independently selected from the group        consisting of hydrogen, a C₁-C₆ alkyl, a halogen, a CYCLIC        SUBSTITUENT, -OR⁸, -SR⁸, -(NR⁸R⁹), TAIL; BRIDGE, -L-Rx and        -L-Sc; where R⁸ and R⁹ are independently a C₁-C₆ alkyl group,        1-2 alicyclic or aromatic rings; or R⁸ and R⁹ taken in        combination are —(CH₂)₂—V—(CH₂)₂- where V is a single bond, —O—,        —CH₂—, or —NR¹⁰-, where R¹⁰ is H or an alkyl having 1-6 carbons;        -   wherein CYCLIC SUBSTITUENT is a substituted or unsubstituted            aryl, heteroaryl or C₃-C₁₀ cycloalkyl;    -   or R⁶ and R⁷ form a fused aromatic ring —R¹¹═R¹²—R¹³═R¹⁴—        wherein R¹¹, R¹², R¹³, and R¹⁴ are independently selected from        the group consisting of hydrogen, C₁-C₆ alkyl group, —OR⁸, —SR⁸,        —(NR⁸R⁹), a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc;    -   R⁵ is independently selected from the group consisting of a        C₁-C₆ alkyl group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and        -L-Sc; or R⁵ is absent;    -   R³⁰, R³¹, and R³² are independently selected from the group        consisting of hydrogen, C₁-C₆ alkyl, C₃-C₁₀ cycloalkyl, aryl,        and heteroaryl, wherein n=0, 1 or 2;    -   wherein, BRIDGE, when present, is bound to a compound having        formula I or another unsymmetrical cyanine dye.

Dyes that are useful for detecting poly(amino acids) are typicallyneutral in charge wherein R⁶ is absent or R² is a C₂-C₆ sulfoalkylgroup. Alternatively, charged dyes wherein X is O or S, n is 0 or 1, R⁶and R⁷ form a fused aromatic ring —R¹¹═R¹²—R¹³═R¹⁴— wherein R¹¹, R¹²,R¹³, and R¹⁴ are independently selected from the group consisting ofhydrogen, C₁-C₆ alkyl group, —OR⁸; and R⁴ and R⁵ are independently aC₁-C₈ alkyl group or a CYCLIC SUBSTITUENT are also employed, andpreferred, for the detection of poly(amino acids).

Dyes that are useful for nucleic acid detection preferably have thefollowing properties wherein X is O, n is 0, R² is a methyl group, R⁶and R⁷ form a fused aromatic ring —R¹¹═R¹²—R¹³═R¹⁴— wherein R¹¹, R¹²,R¹³, and R¹⁴ are independently selected from the group consisting ofhydrogen, C₁-C₆ alkyl group, —OR⁸, and R⁵ and R⁴ are independently aCYCLIC SUBSTITUENT that is an aryl group or an alkyl group.

In one aspect of the invention, that is preferred for detecting nucleicacids, the present dyes comprise at least one TAIL substituent that hasthe formula LINK-SPACER-CAP. LINK is single covalent bond, an eitherlinkage (—O—), a thioether linkage (—S—) or an amine linkage (—NR²⁰—);where R²⁰ is hydrogen, C₁-C₈ alkyl or SPACER-CAP; SPACER is a covalentlinkage; and, CAP is —O—R²¹, —S—R²¹, —NR²¹R²² or —NR²¹R²²R²³; where R²¹,R²², and R²³ are independently selected from the group consisting ofhydrogen, C₁-C₈ alkyl and a C₁-C₈ cycloalkyl wherein said alkyl orcycloalkyl are optionally substituted by one or more substituentsselected from the group consisting of halogen, hydroxyl, C₁-C₈ alkoxy,amino, carboxy, sulfo and phenyl where said phenyl is optionallysubstituted by one or more substituents selected from the groupconsisting halogen, hydroxyl, C₁-C₈ alkoxy, amino, C₁-C₈ aminoalkyl,C₁-C₈ sulfoalkyl and C₁-C₈ carboxyalkyl; or one or more R²¹, R²², andR²³, taken in combination with R²⁰ and SPACER, or with SPACER alone,forms a 5- or 6-membered ring.

In this instance, R⁴ or R⁵ is independently a TAIL or a CYCLICSUBSTITUENT substituted by TAIL. Thus, In one aspect of the invention,R⁴ is a TAIL or a CYCLIC SUBSTITUENT substituted by TAIL and R⁵ is analkyl group wherein CAP is —NR²¹R²² where R²¹ and R²² are independentlyC₁-C₆ alkyl groups. Alternatively, CAP is —NR²¹R²²R²³ where R²¹, R²² andR²³ are independently C₁-C₆ alkyl groups. In another aspect, R⁵ is aCYCLIC SUBSTITUENT that is an aryl or heteroaryl and R⁴ is a TAIL whereCAP is —O—R²¹, —S—R²¹. In yet another aspect, R⁵ is a TAIL, wherein CAPis -NR²¹R²²R²³ where R²¹, R²² and R²³ are independently C₁-C₆ alkylgroups.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows preferential fluorescent staining of double-stranded DNAby Compound 22 in the presence of single-stranded DNA and RNA, asdescribed in Example 35.

FIG. 2: Shows preferential fluorescent staining of double-stranded DNAby Compound 16 in the presence of single-stranded DNA and RNA, asdescribed in Example 35.

FIG. 3: Shows detection of bovine serum albumin using Compound 51, asdescribed in Example 38.

FIG. 4: Shows detection of bovine serum albumin using Compound 44, asdescribed in Example 38.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a fusion protein” includes aplurality of proteins and reference to “a fluorescent compound” includesa plurality of compounds and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the invention as described herein.

The term “alkyl” as used herein refers to a straight, branched or cyclichydrocarbon chain fragment containing between about one and about twentyfive carbon atoms (e.g. methyl, ethyl and the like). Straight, branchedor cyclic hydrocarbon chains having eight or fewer carbon atoms willalso be referred to herein as “lower alkyl”. In addition, the term“alkyl” as used herein further includes one or more substitutions at oneor more carbon atoms of the hydrocarbon chain fragment. Suchsubstitutions include, but are not limited to: aryl; heteroaryl;halogen; alkoxy; amine (—NR′R″); carboxy and thio.

The term “amino” or “amine group” refers to the group —NR′R″ (orN′RR′R″) where R, R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substitutedheteroaryl. A substituted amine being an amine group wherein R′ or R″ isother than hydrogen. In a primary amino group, both R′ and R″ arehydrogen, whereas in a secondary amino group, either, but not both, R′or R″ is hydrogen. In addition, the terms “amine” and “amino” caninclude protonated and quaternized versions of nitrogen, comprising thegroup —N⁺RR′R″ and its biologically compatible anionic counterions.

The term “attachment site” as used herein refers to a site on a moietyor a molecule, e.g. a quencher, a fluorescent dye, an avidin, or anantibody, to which is covalently attached, or capable of beingcovalently attached, to a linker or another moiety.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “—Sc” or “conjugated substance” as used herein refers to abiomolecule or non-biomolecule substance that contains or is modified tocontain a reactive group wherein the reactive group will form a covalentbond with the present compounds that contain an appropriate reactivegroup. A molecule capable of being conjugated to the present compounds,conjugated substance, includes, but not limited to, an amino acid, apeptide, a protein, a polysaccharide, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, ahormone, a lipid, a lipid assembly, a synthetic polymer, a polymericmicroparticle, a biological cell or a virus.

The term “detectable response” as used herein refers to a change in oran occurrence of, a signal that is directly or indirectly detectableeither by observation or by instrumentation. Typically, the detectableresponse is an optical response resulting in a change in the wavelengthdistribution patterns or intensity of absorbance or fluorescence or achange in light scatter, fluorescence lifetime, fluorescencepolarization, or a combination of the above parameters.

The term “dye” as used herein refers to a compound that is fluorescentupon binding or association with an analyte of interest, e.g. nucleicacid or poly(amino acid). These dyes include the present dyes and onesthat can be employed as additional detection reagents. The additionaldetection reagents include fluorophores the are fluorogenic and onesthat inherently fluorescent. Numerous fluorophores are known to thoseskilled in the art and include, but are not limited to, coumarin,acridine, furan, indole, borapolyazaindacene and xanthenes includingfluoroscein, rhodamine, rhodol, cyanine, benzofuran, quinazolinone, andbenzazole as well as other fluorophores described in RICHARD P.HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCHCHEMICALS (9^(th) edition, CD-ROM, 2002).

The term “Linker”, “L” or “BRIDGE” as used herein refers to a covalentlinkage that is a single covalent bond or a series of stable covalentbonds incorporating 1-20 nonhydrogen atoms selected from the groupconsisting of C, N, O, S and P that covalently attach the presentquenching compounds to another moiety such as a chemically reactivegroup or a conjugated substance including biological and non-biologicalsubstances. A “cleavable linker” is a linker that has one or morecovalent bonds that may be broken by the result of a reaction orcondition. For example, an ester in a molecule is a linker that may becleaved by a reagent, e.g. sodium hydroxide, resulting in acarboxylate-containing fragment and a hydroxyl-containing product.

The term “poly(amino acids)” as used herein refers in a generic sense toproteins and polypeptides that include polymers of amino acid residuesof any length. The term “peptide” is used herein to refer topolypeptides having less than 250 amino acid residues, typically lessthan 100 amino acid residues. The terms apply to amino acid polymers inwhich one or more amino acid residues are an artificial chemicalanalogue of a corresponding naturally occurring amino acid, as well asto naturally occurring amino acid polymers.

The term “Rx” or “reactive group” as used herein refers to a group thatis capable of reacting with another chemical group to form a covalentbond, i.e. is covalently reactive under suitable reaction conditions,and generally represents a point of attachment for another substance.The reactive group is a moiety, such as malemide or succinimidyl ester,on the compounds of the present invention that is capable of chemicallyreacting with a functional group on a different compound to form acovalent linkage. Reactive groups generally include nucleophiles,electrophiles and photoactivatable groups.

The term “sample” as used herein refers to any material that may containan analyte of interest, which typically includes poly(amino acids) andnucleic acids. Typically, the sample is a live cell, a biological fluidthat comprises endogenous host cell proteins, nucleic acid polymers,nucleotides, oligonucleotides, peptides and buffer solutions. The samplemay be in an aqueous solution, a viable cell culture or immobilized on asolid or semi solid surface such as a polymeric gel, membrane blot or ona microarray.

II. Compositions and Methods of Use

The invention comprises unsymmetrical cyanine dyes that incorporate anaza-benzazolium ring moiety. Included in the invention are monomeric anddimeric cyanine dyes, including those substituted by a cationic sidechain, a chemically reactive functional group, and a conjugatedsubstance. The dyes of the invention are virtually non-fluorescent whendiluted in aqueous solution but when associated with nucleic acidpolymers such as DNA or RNA, however, the resultant dye-nucleic acidcomplex is extremely fluorescent upon illumination. Similarly, whenassociated with lipid-complexed poly(amino acids), the resultingcomposition is extremely fluorescent upon illumination. The present dyesof the invention stain an analyte of interest, nucleic acids andpoly(amino acida), in a wide variety of samples, particularly in aqueoussolutions, electrophoretic gels, and a wide variety of cells, includingmicroorganisms.

A. Aza-Benzazolium Containing Cyanine Dyes

The dyes of the invention comprise a cyanine dye that contains: 1) afirst heterocyclic ring system that is a substituted aza-benzazoliumring, 2) a bridging methine and 3) a second heterocyclic ring systemthat is a pyridine, a quinoline, a pyridinium or a quinolinium. In oneembodiment of the invention, the first or second ring system issubstituted by a side chain, TAIL, that contains at least oneheteroatom. In another embodiment, the first or second ring system iscovalently bound to a second cyanine dye, forming a homo- orheterodimer. The first and second ring systems of the first or secondcyanine dye is optionally further substituted by a variety ofsubstituents, including chemically reactive groups as described below.

In one embodiment these dyes are pyridinium and quinolinium-based dyes,wherein the dye has the following formula:

-   -   wherein A represents the atoms necessary to form one to two        fused aromatic rings having 6 atoms in each ring, at least one        of which is a nitrogen atom, said ring or rings being optionally        further substituted one or more times by C₁-C₂₂ alkyl, C₁-C₂₂        alkoxy trifluoromethyl, halogen, BRIDGE, -L-Rx or -L-Sc;        -   wherein Rx is a reactive group; Sc is a conjugated            substance; and L and BRIDGE are independently a single            covalent bond, or a covalent linkage;    -   X is O, S, Se, NR¹⁵, or CR¹⁶R¹⁷, where R¹⁵ is H or an alkyl        group having 1-22 carbons; and R¹⁶ and R¹⁷, which may be the        same or different, are independently alkyl groups having 1-22        carbons, or R¹⁶ and R¹⁷ taken in combination complete a five or        six membered saturated ring;    -   R² is selected from the group consisting of -L-Rx, -L-Sc, TAIL,        BRIDGE and an alkyl group having 1-22 carbons that is optionally        substituted by sulfo, carboxy, amino, substituted amino or        substituted ammonium, wherein α is 0 or 1; and TAIL is a        heteroatom-containing moiety;    -   Y is —CR³═CR⁴— wherein p and m=0 or 1, such that p+m=1;    -   R³, R⁴, R⁶, and R⁷ are independently selected from the group        consisting of hydrogen, a C₁-C₂₂ alkyl, a halogen, a CYCLIC        SUBSTITUENT, —OR⁸, —SR⁸, —(NR⁸R⁹), TAIL; BRIDGE, -L-Rx and        -L-Sc; where R⁸ and R⁹ are independently a C₁-C₆ alkyl group,        1-2 alicyclic or aromatic rings; or R⁸ and R⁹ taken in        combination are —(CH₂)₂—V—(CH₂)₂— where V is a single bond, —O—,        —CH₂—, or —NR¹⁰—, where R¹⁰ is H or an alkyl having 1-6 carbons;        -   wherein CYCLIC SUBSTITUENT is a substituted or unsubstituted            aryl, heteroaryl or C₃-C₁₀ cycloalkyl;    -   or R⁶ and R⁷ form a fused aromatic ring —R¹¹═R¹²—R¹³═R¹⁴—        wherein R¹¹, R¹², R¹³, and R¹⁴ are independently selected from        the group consisting of hydrogen, C₁-C₂₂ alkyl group, —OR⁸,        —SR⁸, —(NR⁸R⁹), a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and        -L-Sc;    -   R⁵ is independently selected from the group consisting of a        C₁-C₂₂ alkyl group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx        and -L-Sc; or R⁵ is absent;    -   R³⁰, R³¹, and R³² are independently selected from the group        consisting of hydrogen, C₁-C₂₂ alkyl, C₃-C₁₀ cycloalkyl, aryl,        and heteroaryl, wherein n=0, 1 or 2;    -   wherein, BRIDGE, when present, is bound to a compound having        formula I or another unsymmetrical cyanine dye.

The A moiety represents the atoms necessary to form one or more fused6-membered aromatic rings, incorporating at least one nitrogen atom═N(R²)_(β)—. The six membered rings of the aza-benzazole ring systemtypically incorporate 1-3 nitrogen atoms, more preferably 1-2 nitrogenatoms. The nitrogen atoms are typically incorporated in the first6-membered aromatic ring fused to the azole ring. Preferred embodimentsof the aza-benzazole moiety include without limitation the followingstructures,

At least one of the aza-benzazole nitrogen atoms is quaternized by thesubstituent R², resulting in a formal positive charge. In oneembodiment, α=1 and β=0, such that the azole nitrogen atom isquaternized, and the benzo nitrogen atom is unsubstituted. In anotherembodiment, α=0 and β=1, such that the azole nitrogen atom isunsubstituted and at least one benzo nitrogen atom is quaternized by R².Either α or a β is 1, and typically no more than one β in a givenaza-benzazole is 1. The presence of an additional nitrogen atomincorporated in the fused aromatic ring of the benzazole moiety resultsin a shift of the emission spectra to longer wavelength. The presence ofadditional nitrogen atoms (as in structure V, above) shift thewavelength even further, as does the presence of additional fused6-membered rings.

The aza-benzazole moiety is optionally further substituted at one ormore aromatic carbons by an alkyl group having from 1-6 carbons, alkoxyhaving from 1-6 carbons, trifluoromethyl, halogen, BRIDGE, -L-Rx or-L-Sc. Although the carbon atoms of the aza-benzazolium ring system aretypically fully substituted by hydrogen, incorporation of one or morenon-hydrogen substituents can be used to fine tune the absorption andemission spectrum of the resulting dye. Typically, where present, thereis only one non-hydrogen substituent on the aza-benzazole, preferably analkoxy or halogen substituent.

R_(x) is a reactive group that provides compounds of the presentinvention that can be covalently attached to another substance, aconjugated substance, such as a biomolecule or non-biomolecule includingproteins and non-protein molecules such as dextran and a microparticle.The reactive group is typically a nucleophile, electrophile orphotoactivatable group that chemically reacts with another appropriatereactive group on a biomolecule or non-biomolecule to form a covalentbond between the compounds of the present invention and anothersubstance. Thus, a conjugated substance (S_(c)) can be any molecule withan appropriate reactive group, or is modified to contain an appropriatereactive group, that can be covalently bonded to the compounds of thepresent invention.

L is a covalent linkage that attaches the reactive group or conjugatedsubstance to the present compounds wherein the covalent linkage may be asingle covalent bond or a series of stable chemical bonds containing1-20 nonhydrogen atoms. These atoms are selected from the groupconsisting of C, N, P, O or S. As used herein the term “BRIDGE” is alsoa covalent linkage having the same properties as L, but that attachesany one of the present dyes to another dye to form a homo- orhetero-dimer.

The substituent R² is an alkyl group having 1-22 carbons, that is linearor branched, saturated or unsaturated, that is optionally substitutedone or more times by hydroxy, carboxy, sulfo, amino, amino substitutedby 1-2 C₁-C₆ alkyls, or ammonium substituted by 1-3 C₁-C₆ alkyls. The R²substituent is alternatively a TAIL moiety, BRIDGE, a reactive group(-L-Rx) or a conjugated substance (-L-Sc). In one embodimentparticularly suited for staining nucleic acids, R² is alkyl having 1-6carbons, preferably methyl or ethyl, more preferably methyl. In anotherembodiment particularly suitable for staining lipid-complexed poly(aminoacids), R² is a sulfoalkyl having 2-6 carbons.

As used herein the term “TAIL” refers to a heteroatom-containing sidechain that is described by the formula LINK-SPACER-CAP. LINK is thelinking moiety by which TAIL is attached to the dyes of the presentinvention. SPACER is a covalent linkage that connects LINK to CAP. CAPis the portion of TAIL that possesses a heteroatom component. Where thedye is substituted by a TAIL moiety, typically one or more of R², R³,R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³ and R¹⁴ is a TAIL. In one embodiment ofthe invention, at least one of R³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³ and R¹⁴is required to be a TAIL. Preferably, R⁴ or R⁵ is a TAIL or aTAIL-substituted CYCLIC SUBSTITUENT. When R⁴ is a TAIL, LINK ispreferably —NR²⁰— or —S—. When TAIL is at any position other than R⁴ orR⁵, LINK is preferably —O— or a single bond.

As used herein the term “CYCLIC SUBSTITUENT” refers to a substituentthat is a substituted or unsubstituted aryl, heteroaryl, or cycloalkylhaving 3-10 carbons. As used herein, an aryl is a phenyl or a naphthylgroup, and a heteroaryl substituent is a 5 or 6-membered heteroaromaticring, wherein the heteroatom is O, N or S. The CYCLIC SUBSTITUENT isoptionally substituted by halogen, amino, alkyl, perfluoroalkyl,alkylamino, dialkylamino, alkoxy or carboxyalkyl, wherein each alkylgroup has 1-6 carbons, or by a TAIL moiety. The CYCLIC SUBSTITUENT ispreferably a substituted or unsubstituted naphthyl, phenyl, thienyl, orcycloalkyl having 3-10 carbons, more preferably, the CYCLIC SUBSTITUENTis phenyl.

Where the dye is substituted by a CYCLIC SUBSTITUENT, typically one ofR³, R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³ and R¹⁴ is a CYCLIC SUBSTITUENT.Preferably, one or more of R⁴, R⁵, R⁶, or R¹² is TAIL or a CYCLICSUBSTITUENT, including CYCLIC SUBSTITUENTS that are substituted by TAILmoieties.

In one embodiment, X is one of O, S, Se or NR¹⁵, where R¹⁵ is H or analkyl group having 1-22 carbons, that is linear or branched, saturatedor unsaturated, that is optionally substituted one or more times byhydroxy, carboxy, sulfo, amino, amino substituted by 1-2 C₁-C₆ alkyls,or ammonium substituted by 1-3 C₁-C₆ alkyls. The R¹⁵ substituent isalternatively a TAIL moiety.

Alternatively, X is CR¹⁶R¹⁷, where R¹⁶ and R¹⁷, which may be the same ordifferent, are independently H or alkyl groups having 1-22 carbons, thatare linear or branched, saturated or unsaturated, that are optionallysubstituted one or more times by hydroxy, carboxy, sulfo, amino, aminosubstituted by 1-2 C₁-C₆ alkyls, or ammonium substituted by 1-3 C₁-C₈alkyls. Alternatively, one of R¹⁶ and R¹⁷ is a TAIL moiety.Alternatively, the carbons of R¹⁶ and R¹⁷ taken in combination completea five or six membered saturated ring. When X is CR¹⁶R¹⁷, R¹⁶ and R¹⁷are typically alkyls having 1-6 carbons, preferably methyl or ethyl.

Preferably, X is O or S, more preferably X is S.

The two heterocyclic ring systems of the cyanine dye are linked by abridging methine having the formula—(CR³²═CR³¹)_(n)—CR³⁰═

(where n=0, 1, or 2) in such a way as to permit extensive electronicdelocalization. When n =0 the dyes are unsymmetrical monomethine dyes;when n=1 the dyes are trimethine dyes; when n=2, the dyes arepentamethine dyes. As with similar compounds, the number of methinegroups between the heteroaromatic rings influences the spectralproperties of the dye. The monomethine dyes of the present inventiontypically have yellow to orange to red fluorescence emission. Thetrimethine dye analogs are substantially shifted toward red wavelengthsbeyond 600 nm, and the pentamethine dyes are shifted even further, oftenexhibiting infrared fluorescence emission. Preferably n=0 or 1, morepreferably n=0.

Methine substituents R³⁰, R³¹, and R³² are independently H, alkyl having1-6 carbons, cycloalkyl having 3-10 carbons, aryl, or heteroaryl. Wheren=1 or 2, each R³¹ and R³² varies independently. In one embodiment, onlythe substituent on the central carbon of the methine bridge isnonhydrogen (R³⁰ where n=0, R³¹ where n=1, the centrally located R³²where n=2). In another embodiment, where a methine substituent isnonhydrogen it is an alkyl. In another embodiment, where a methinesubstituent is nonhydrogen it is an aryl. Typically, each R³⁰, R³¹, andR³² is hydrogen. Where R³⁰, R³¹, or R³² is nonhydrogen, preferably n=1.

In one aspect of the invention, selected cyanine dyes having nonhydrogenmethine substituents have particular utility as RNA selective nucleicacid stains. In another aspect of the invention, cyanine dyes havingnonhydrogen methine substituents increase the signal-to-noise ratio whenstaining nucleic acids by decreasing background fluorescence.

The incorporation of substituted methine bridges, as described above,also provides useful derivatives of previously described cyanine dyes(e.g., U.S. Pat. No. 5,321,130 to Yue et al. (1994); U.S. Pat. No.5,410,030 to Yue et al. (1995); U.S. Pat. No. 5,436,134 to Haugland etal. (1995); U.S. Pat. No. 5,582,977 to Yue et al. (1996); U.S. Pat. No.5,658,751 to Yue et al. (1997); and U.S. Pat. No. 5,863,753 to Hauglandet al. (1999)).

The second ring system contains a ring fragment Y that is —CR³═CR⁴—,with subscripts p and m equal to 0 or 1, such that p+m=1. The secondring system is a 6 membered heterocycle according to one of theseformulations

In preferred embodiments of the invention, m=1 and p=0 (“4-pyridiniums”and “4-quinoliniums”).

The pyridinium and quinolinium-based cyanine dyes include anN-substituent R⁵ that is an alkyl that is saturated or unsaturated,linear or branched, having 1-22 carbons, that is optionally substitutedone or more times by hydroxy, carboxy, sulfo, amino, amino substitutedby 1-2 C₁-C₆ alkyls, or ammonium substituted by 1-3 C₁-C₆ alkyls.Alternatively, R⁵ is a CYCLIC SUBSTITUENT, a TAIL moiety, BRIDGE, areactive group, a conjugated substance, a pair of electrons, orsulfoalkyl. Typically R⁵ is an alkyl having 1-6 carbons, preferably 1-2carbons, or R⁵ is a CYCLIC SUBSTITUENT. In one embodiment, R⁵ is asulfoalkyl having 2-6 carbons. In another embodiment, R⁵ is absent andreplaced by a pair of electrons, resulting in a neutral unsymmetricalcyanine dye, as described below.

The substituents of the second ring system, R³, R⁴, R⁶, and R⁷, areindependently H, halogen, an alkyl that is saturated or unsaturated,linear or branched, having 1-6 carbons, a CYCLIC SUBSTITUENT, a TAILmoiety, BRIDGE a reactive group (Rx) or a conjugated substance (Sc). R³,R⁴, R⁶, and R⁷ are optionally —OR⁸, —SR⁸, or —(NR⁸R⁹) where R⁸ and R⁹,which can be the same or different, are independently alkyl groupshaving 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R⁸ and R⁹taken in combination form a cyclic structure having the formula—(CH₂)₂—W—(CH₂)₂—, where W is a single bond, —O—, —CH₂—, or —NR¹⁰—,where R¹⁰ is H or an alkyl having 1-6 carbons. In addition, R³ and R⁴are optionally and independently —OSO₂R¹⁹ where R¹⁹ is alkyl having 1-6carbons, or perfluoroalkyl having 1-6 carbons, or aryl.

Optionally, R⁶ and R⁷ taken in combination are —(CH₂)_(v)— where v=3 or4, forming a fused 5- or 6-membered ring, or R⁶ and R⁷, taken incombination form a fused 6-membered aromatic ring.

Alternatively, any of R³, R⁴, R⁶ or R⁷ is a CYCLIC SUBSTITUENT.Preferred ring substituents are independently H, alkyl, —OR⁸, or aCYCLIC SUBSTITUENT, or a TAIL. In one aspect of the invention, R⁴ is nothydrogen. In another aspect of the invention, R⁴ is a CYCLICSUBSTITUENT, a TAIL moiety, or a CYCLIC SUBSTITUENT that is furthersubstituted by a TAIL moiety.

Where R⁶ and R⁷ taken in combination form a fused 6-membered aromaticring, embodiments of this invention are quinolinium derivatives, and thesecond ring system has the formula

where ring substituents R¹¹, R¹², R¹³, and R¹⁴ are independently H, analkyl that is saturated or unsaturated, linear or branched, having 1-6carbons; halogen, —OR⁸, —SR⁸, —(NR⁸R⁹), where R⁸ and R⁹ are as definedpreviously; or a CYCLIC SUBSTITUENT, a TAIL moiety, BRIDGE, a reactivegroup or a conjugated substance. Typically no more than one of R¹¹, R¹²,R¹³, and R¹⁴ is nonhydrogen, and is preferably halogen or —OR⁸.Preferred embodiments of the invention are quinoliniums wherein m=1 andp=0 (“4-quinoliniums”). The presence of the fused 6-membered ring at R⁶and R⁷ typically increases the wavelength of the dye's fluorescence whenassociated with nucleic acids or lipid-complexed poly(amino acids).

In another embodiment of the invention, the dye of the invention is ahomodimer or heterodimer of unsymmetrical cyanine dyes. That is, twocyanine dye subunits, at least one of which has a structure as describedabove, are joined by a covalent linkage, BRIDGE, to yield a dimericcompound having utility as a fluorescent stain for nucleic acids orlipid-complexed poly(amino acids) (see Compound 28, Example 12 andCompound 46, Example 13). While one cyanine dye subunit is anaza-benzazolium based cyanine dye subunit, the other portion of thedimeric dye may be a cyanine dye as described previously by Haugland andYue (U.S. Pat. No. 5,321,130; U.S. Pat. No. 5,436,134; U.S. Pat. No.5,658,751; and U.S. Pat. No. 5,869,689). Alternatively, the otherportion of the dimeric dye is dissimilar in structure, for example aphenanthridium moiety.

The BRIDGE moiety is typically a combination of atoms having stablechemical bonds. The BRIDGE moiety optionally includes single, double,triple or aromatic carbon-carbon bonds, as well as carbon-nitrogenbonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfurbonds. The BRIDGE moiety typically includes ether, thioether,carboxamide, sulfonamide, urea, urethane or hydrazine moieties.Preferred BRIDGE moieties have 1-20 nonhydrogen atoms selected from thegroup consisting of C, N, O and S; and are composed of any combinationof ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazidebonds and aromatic or heteroaromatic bonds. Preferably BRIDGE is acombination of single carbon-carbon bonds and carboxamide or thioetherbonds. Examples of BRIDGE include substituted or unsubstitutedpolymethylene, arylene, alkylarylene or arylenealkyl.

In a preferred embodiment, the BRIDGE moiety is an aliphatic chaincontaining a backbone of 4-19 carbon atoms. The carbon backbone may beinterspersed at one or more intervals with a non-carbon heteroatom. Theheteroatoms, which may be the same or different, are N, O, or S,preferably the heteroatom is N. Where the heteroatom is nitrogen, it isoptionally substituted with one or more alkyl substituents having 1-6carbon atoms, which may be the same or different. BRIDGE moieties thatincorporate quaternary nitrogens confer cationic charges on the dimersof the invention, potentially increasing their affinity for nucleicacids, as well as decreasing their ability to permeate cell membranes.

Where the dimeric dye incorporates two cyanine subunits, the BRIDGEmoiety is independently bound to each cyanine subunit at one of R², R³,R⁴, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, or R¹⁴, as defined above. Preferably theBRIDGE moiety is independently bound to each cyanine subunit at R², R⁴or R⁵, more preferably at R⁴ or R⁵.

By careful selection of the cyanine subunits that are covalently joinedto form the dimeric dye, a variety of dimers with desirable spectralproperties may be prepared. Selection of subunits that function as anenergy donor and energy acceptor, respectively, via fluorescenceresonance energy transfer typically produces a dimeric stain with anenhanced Stokes shift. The necessary difference in spectral propertiesfor energy transfer to occur may be created by covalently attaching anaza-benzothiazole cyanine subunit to an aza-benzoxazole cyanine subunit.Alternatively, a monomethine cyanine subunit may be attached to atrimethine or pentamethine cyanine subunit, or a cationic cyaninesubunit may be attached to a neutral cyanine dye subunit. In anotheraspect of the invention, an aza-benzoxazole cyanine subunit is attachedto a non aza-substituted cyanine subunit. In yet another aspect of theinvention, an aza-benzoxazole cyanine subunit is attached to anon-cyanine subunit, typically a phenanthridium subunit. Dimeric dyesincorporating non-aza-benzazolium containing cyanine subunits havepreviously been described (U.S. Pat. No. 5,582,977 to Yue et al. (1996);U.S. Pat. No. 5,401,847 to Glazer, (1995); U.S. Pat. No. 5,565,554 toGlazer (1996); and U.S. Pat. No. 5,760,201 to Glazer et. al. (1998)).

In a specific embodiment of the invention, the dyes of the invention are4-pyridiniums or 4-quinoliniums, wherein R⁵ is an alkyl having 1-6carbons, or R⁵ is a CYCLIC SUBSTITUENT, and R⁴ is not hydrogen. Dyeshaving an R⁴ substituent that is non-hydrogen typically possess enhancedquantum yields relative to similar dyes wherein R⁴ is H. For this classof dyes, R⁵ is preferably alkyl having 1-6 carbons. Preferably R⁴ is aTAIL.

In an additional preferred embodiment of the invention, the secondheterocyclic ring contains exactly two non-hydrogen substituents, one ofwhich is a TAIL.

Some of the dyes of the present invention that possess a TAIL moiety atR⁴ or R⁵ exhibit particular utility for staining nucleic acids in cellsand microorganisms. The utility of specific embodiments of the dyes ofthe present invention in staining cells and microorganisms is generallydependent on the chemical nature of the TAIL moiety, and the identity ofthe group present at R⁵. For example, those compounds for which CAP is aquaternary ammonium salt are generally impermeant to living cells, withthe exception of some yeast cells. However, the permeability of thosecompounds for which CAP is a primary or secondary amine, and LINK is asecondary or tertiary amine, is typically related to the nature of R⁵(the N-substituent).

Typically, dyes useful as impermeant cellular probes are those dyeshaving 2 or more positive charges overall, more preferably 3 or morepositive charges. Preferred dyes for permeant cellular probes are dyeswherein R⁵ is aryl or heteroaryl and CAP is —O—R²¹ or —S—R²¹, or dyeswherein R⁴ is a CYCLIC SUBSTITUENT substituted by a cationic TAIL (forexample, Compound 24, Example 10).

Dyes that are preferred for staining nucleic acids present inelectrophoretic gels typically have a CAP that is a dialkylamino group,while dyes useful as stains for poly(amino acids) in electrophoreticgels are overall neutral in charge, typically by virtue of a sulfoalkylgroup present at R² or R⁵ that balances the cationic charge on theaza-benzazole moiety.

Any net positive or negative charges possessed by the dyes of theinvention are balanced by a necessary counterion or counterions.Typically, the dyes of the invention are positively charged due to thepresence of a quaternized aza-benzazole nitrogen, however the overallcharge on the dye is determined by the charge present on each dyesubstituent (such as a negatively charged sulfonic acid group, or apositively charged ammonium group). Where necessary, the counterion isdepicted as ψ and the polarity of the charge is indicated. Examples ofuseful counterions for dyes having a net positive charge include, butare not limited to, chloride, bromide, iodide, sulfate, alkanesulfonate,arylsulfonate, phosphate, perchlorate, tetrafluoroborate,tetraarylboride, nitrate and anions of aromatic or aliphatic carboxylicacids. Preferred negative counterions are chloride, iodide, perchlorateand various sulfonates. Examples of useful counterions for dyes having anet negative charge include, but are not limited to, alkali metal ions,alkaline earth metal ions, transition metal ions, ammonium orsubstituted ammonium ions. Preferably, any necessary counterion isbiologically compatible, is not toxic as used, and does not have asubstantially deleterious effect on biomolecules.

In one embodiment, the cyanine dyes of the invention are substituted byat least one TAIL moiety having the formula LINK-SPACER-CAP.

LINK is a single covalent bond, an ether linkage (—O—), a thioetherlinkage (—S—), or an amine linkage (—NR²⁰—). In each embodiment, LINKforms the attachment between the cyanine dye core structure and SPACER.When LINK is an amine, the amine substituent (R²⁰) is optionally H, suchthat LINK=—NH—. Alternatively, R²⁰ is a linear or branched alkyl having1-8 carbons. In another embodiment of the invention, R²⁰ is—SPACER′-CAP′, yielding a TAIL having the formula

where SPACER′ and CAP′, respectively, may be the same as or differentfrom SPACER and CAP, and are selected from the same alternatives definedfor SPACER and CAP, respectively. For the sake of simplifying thedescription, SPACER and CAP are defined with the understanding that adescription of SPACER includes SPACER′, and a description of CAPincludes CAP′.

SPACER is a covalent linkage that joins LINK and CAP. SPACER is alinear, branched, cyclic, heterocyclic, saturated or unsaturatedarrangement of 1-16 C, N, P, O or S atoms. Alternatively, SPACER is asingle covalent bond, such that both LINK and SPACER are notsimultaneously single covalent bonds. Preferably, the SPACER linkagemust begin and end with a carbon atom. Typically, if SPACER consists ofa single atom, it is required to be a carbon atom, so that the first andlast atom in SPACER (in this specific instance, they are the same atom)is a carbon. The 1-16 atoms making up SPACER are combined using anyappropriate combination of ether, thioether, amine, ester, or amidebonds; or single, double, triple or aromatic carbon-carbon bonds; orphosphorus-oxygen bonds; or phosphorus-sulfur bonds; ornitrogen-nitrogen bonds; or nitrogen-oxygen bonds; or aromatic orheteroaromatic bonds. SPACER is further substituted by hydrogen toaccommodate the valence state of each atom in SPACER.

Generally, the atoms of SPACER are arranged such that all heteroatoms inthe linear backbone of SPACER are separated by at least one carbon atom,and preferably separated by at least two carbon atoms. Typically, SPACERis 1-6 carbon atoms in a linear or branched saturated chain. In oneembodiment of the invention, SPACER incorporates a 6-membered aromaticring (phenylene linkage). In another embodiment of the invention, SPACERincorporates a 5- or 6-membered heteroaromatic ring, wherein theheteroatoms are O, N, or S. Alternatively, SPACER incorporates amidelinkages, ester linkages, simple ethers and thioethers, and amines in alinear arrangement, such as —CH₂—CH₂—(C═O)—NH—CH₂—CH₂—CH₂—. Preferably,SPACER is an alkylene (—(CH₂)_(k)—, where k=1-8).

LINK and SPACER, in combination, serve to attach a heteroatom-containinggroup, CAP, to the dye core structure. CAP may contain oxygen, sulfur ornitrogen, according to the formulas —O—R²¹, —S—R²¹, —NR²¹R²², or—NR²¹R²²R²³. The substituents R²¹, R²², and R²³ are independently H, ora linear or branched alkyl or cycloalkyl having 1-8 carbons. Where anyof R²¹, R²² and R²³ are alkyl or cycloalkyl, they are optionally furthersubstituted by one or more halogen, hydroxy, alkoxy having 1-8 carbons,amino, carboxy, sulfo, or phenyl, where phenyl is optionally furthersubstituted by halogen, hydroxy, alkoxy having 1-8 carbons, amino,aminoalkyl having 1-8 carbons, or sulfoalkyl or carboxyalkyl having 1-8carbons. In another embodiment of the invention, one or more of R²¹, R²²and R²³, taken in combination with R²⁰ and SPACER, or with SPACER alone,forms a 5- or 6-membered ring that is aromatic, heteroaromatic,alicyctic or heteroalicyclic ring. When the 5- or 6-membered ring isheteroaromatic or heteroalicyclic, the ring contains 1-3 heteroatomsthat are O, N or S. Preferably, R²¹ and R²² are independently hydrogenor alkyls having 1-8 carbons. R²³ is typically H or alkyl having 1-8carbons.

When CAP is —NR²¹R²²R²³, the CAP nitrogen atom is formally positivelycharged, and adds to the cumulative charge that is balanced by thepresence of the counterion ψ.

Additionally, there are several embodiments of the present inventionwherein CAP incorporates a cyclic structure. In these embodiments, CAPtypically incorporates a 5- or 6-membered nitrogen-containing ring,optionally including an additional heteroatom (typically oxygen), wherethe ring nitrogen is optionally substituted by R²³ to give an ammoniumsalt. Specific versions of CAP include, but are not limited to, thoselisted in Table 1.

TABLE 1 Examples of specific CAP moieties

CAP is preferably —NR²¹R²² or —NR²¹R²²R²³, where R²¹, R²², and R²³ arealkyls having 1-6 carbons. More preferably CAP is —N(CH₃)₂ or —N(CH₃)₃.

Preferably TAIL contains 6-10 non-hydrogen atoms, including LINK andCAP.

Selected examples of TAIL are listed in Table 2. For each TAIL, theidentities of LINK, SPACER and CAP are specified. Where R²¹, R²², or R²³combined with either R²⁰ or SPACER, the combination is indicated in thetable.

TABLE 2 Specific examples of TAIL moieties SPACER CAP TAIL LINK SPACER′CAP′

—CH₂—CH₂—CH₂— —N(CH₃)₂

—CH₂—CH₂—CH₂— —N⁺(CH₃)₃

—CH₂—CH₂—CH₂— —CH₂—CH₂—CH₂  —N(CH₃)₂ —N(CH₃)₂

—CH₂—CH₂—CH₂— —CH₂—CH₂—CH₂  —N⁺(CH₃)₃ —N⁺(CH₃)₃

—S— —CH₂—CH₂  —N(CH₃)₂

—S— —CH₂—CH₂  —N⁺(CH₃)₃

—N(—R²²)— —CH₂—CH₂  —N(CH₃) (CH₂—CH₂—R²⁰)

—N(—R²³)— —CH₂—CH₂  —N⁺(CH₃)₂ (CH₂—CH₂—R²⁰)

—S— —C(—R²²) ═N—CH₂ CH₂ CH₂   NH (-SPACER)

bond (p-phenylene)-CH₂— —N⁺(CH₃) (CH₂CH₃)₂

—O— —CH₂—CH₂—CH₂  —N(CH₃)₂

bond (p-phenylene)- O—CH₂—CH₂—CH₂  —N⁺(CH₃)₃

—S—

—OCH₂CH₃

—NH— bond —N(CH₃)₂

—S— —CH₂—CH₂—(C═O) —NH—CH₂—CH₂—CH₂  —N(CH₃)₂

In another embodiment of the invention, the cyanine dyes of theinvention are described by the formula

where A, X, R², α, n, Y_(m), Y_(p), R⁶, R⁷, R³⁰, R³¹, R³², andsubstitution patterns and preferences are as defined previously. These‘free base’ versions of the cyanine dyes of the invention are notderivatized by an R⁵ hydrogen or non-hydrogen atom containingsubstituent, instead a pair of electrons is present at the R⁵ position,wherein the neural compound incorporates an aza-benzazole ring system, amethine moiety, and a pyridine or quinoline ring system. Related neutralcyanine dyes are described in U.S. Pat. No. 5,656,449 to Yue (1997).

B. Synthesis of the Present Dye Compounds

A useful synthetic route to the dyes of the present invention can bedescribed in three parts, following the natural breakdown in thedescription of the compounds. In general, the synthesis of these dyesrequires three precursors: the appropriate azabenzazolium, orpolyazabenzazolium salt; the appropriate pyridine, quinoline, pyridiniumor quinolinium; and (where n=1 or 2) a source for the methine spacer.Typically each component is selected so as to incorporate theappropriate chemical substituents, or functional groups that can beconverted to the appropriate substituents. The chemistry that isrequired to prepare and combine these precursors so as to yield any ofthe subject derivatives is generally well understood by one skilled inthe art (see U.S. Pat. No. 5,436,134 to Haugland et al. (1995); U.S.Pat. No. 5,656,449 to Yue (1997); U.S. Pat. No. 5,658,751 to Yue et al.(1997), U.S. Pat. No. 5,863,753 to Haugland et al. (1999)). Althoughthere are many possible variations that may yield an equivalent result,we provide herein some useful general methods for their synthesis andincorporation of chemical modifications.

1. The Azabenzazollum or Polyazabenzazollum Moiety

A wide variety of derivatives suitable for the preparation of the dyesof the invention have been previously described (see, for example,Brooker, et al., J. AM. CHEM. SOC., 64, 199 (1942); Heravi, et al.,INDIAN J. CHEM. 36B, 1025 (1997); Smith et al. SULFUR LETTERS 17, 197(1994); Chu-Moyer et al. J. ORG. CHEM. 60, 5721 (1995); Turner, J. ORG.CHEM. 48, 3401 (1983); Couture et al. J. HETEROCYCLIC CHEM. 24, 1765(1987); Petric et al. J. HETEROCYCLIC CHEM. 14, 1045, (1977); Barlin etal. AUST. J. CHEM., 37, 1729 (1984); Saikachi et al. CHEM. & PHARM.BULL. 9, 941 (1961); Barlin AUST. J. CHEM. 36, 983 (1983); Foye et al.,J. PHARM. SCI. 64, 1371 (1975); Khanna et al. J. ORG. CHEM. 60, 960(1995)); British Patent No. 870,753 to Ficken et al. (1961); Ficken atal., “Diazaindenes and Their Quaternary Salts-Part I” pp 3202-3212(1959); Ficken at al., “Diazaindenes and Their Quaternary Salts-Part II”pp 584-588 (1961); These synthetic precursors have the common structure:

where A, X, R², and, α, have been defined previously. If X is O, theprecursor compound is an aza- or polyaza-benzoxazolium; if X is S it isan aza- or polyaza-benzothiazolium; if X is Se it is an aza- orpolyaza-benzoselenazollum; if X is N or an alkyl-substituted N it is anaza- or polyaza-benzimidazolium; and if X is CR¹⁶R¹⁷ then it is an aza-or polyaza-indolinium derivative. Commonly R¹⁶ and R¹⁷ are both methyl.However, methods for preparing compounds where R¹⁶ and R¹⁷ are notmethyl are known (Smith at al., SULFUR LETTERS, 18, 79 (1995); Smith etal., CHEM INDUSTRY, 9, 302 (1988); Couture at al. HETEROCYCLES 22, 1383(1984)).

The substituents on the aromatic carbons of the aza-benzazolium moietyare typically incorporated in the parent aza- or polyaza-benzazolemolecule prior to quaternization with an alkylating agent. However, suchsubstituents may also be incorporated during the synthesis of theaza-benzazole moiety (Examples 1-4). R² is usually obtained byalkylation of the parent heterocycle with an alkylating agent R²—Z,where R² is a substituent as described above, in particular where R² isan alkyl group having 1-6 carbons, that is optionally substituted bysulfonate, carboxy, amino, or a chemically reactive group, or aprecursor necessary for formation of a TAIL moiety, or attachment of aBRIDGE precursor, or second dimer subunit. Z of the alkylating agent isan electronegative group that frequently becomes the counterion on theresultant dye, ψ. The counterion may be exchanged for another counterionby methods known in the art, such as the use of ion exchange resins orby precipitation. Selected examples of R²—Z include methyl iodide,diethyl sulfate, hexyl p-toluenesulfonate, sulfopropyl iodide, andsulfobutyliodide. Preferred R²—Z are compounds that yield R²=methyl,such as methyl iodide, methyl methanesulfonate, dimethyl sulfate, methyltrifluoromethanesulfonate or methyl p-toluenesulfonate.

D is a substituent whose nature is determined by the synthetic methodutilized to couple the azabenzazolium or polyazabenzazolium precursorwith the desired precursor of the second heterocyclic ring system. Whenn=0, D is typically alkylthio, commonly methylthio, or D is chloro,bromo or iodo. When n=1 or 2, D is methyl. Only in the case of D=methylis any part of D incorporated in the final compound.

The aza-benzoxazolium precursor compounds wherein X═O have not beenpreviously described.

The preparation of useful intermediates for the synthesis of the dyes ofthe invention via acetylation of a 2-amino-3-hydroxypyridine hassimilarly not been previously described (Examples 5 and 21).

2. The Second Heterocyclic Ring System that is a Pyridine, a Quinoline,a Pyridinium or a Quinolinium

A variety of intermediate compounds useful as precursors for the secondheterocyclic ring system can be used to synthesize the secondheterocyclic ring system; many of which are available to one skilled inthe art. Compounds containing the quinolinium moiety in this inventiondiffer from those that contain a single pyridinium ring only in thepresence of an additional aromatic ring containing four carbon atomsthat is fused at the R⁶ and R⁷ positions of the parent structure. Exceptwhere reference is to a specific pyridine or pyridinium salt, it isunderstood that mention of pyridines or pyridinium salts encompassesbenzopyridines and benzopyridinium salts, which are formally calledquinolines or quinolinium salts. Mention of quinolines and quinoliniumsalts refer only to structures containing at least two fused aromaticrings. Similarly, pyridine and quinoline precursors are not formallycharged, and are not substituted by an R⁵ substituent.

In the synthesis of the dyes of the invention, the second heterocyclicprecursor is usually appropriately substituted. Alternatively,substituents can be incorporated into the precursor structure subsequentto attachment of the azabenzazolium portion of the dye. One of thesubstituents that may be incorporated before or after reaction with theazabenzazolium or polyazabenzazolium precursor is TAIL.

There exist two major structural distinctions within the family of dyesdescribed in the invention that are related to the point of attachmentof the second heterocyclic ring system. In one case (where m=0 and p=1)the position of attachment places the methine moiety adjacent to thering nitrogen (“2-substituted”). In the more common case (where m=1 andp=0) the position of the nitrogen atom is para to the point ofattachment (“4-substituted”).

Typically the required pyridinium salt precursor has the structure

and the quinolinium salt precursor has the general structure

with the substituents as defined previously. At all times, the ring is a6-membered pyridinium-based heterocycle.

When n=0, B is methyl, or B is chloro, bromo or iodo. When n=1 or 2, Bis methyl. Only when n=1 or n=2 is any part of B incorporated in thefinal compound.

There are several general published methods for the synthesis ofderivatives of pyridinium and pyridine, including those derivativeshaving substituents at any available position, including substitutionsthat are TAIL or that can be converted to TAIL before or after reactionwith the azabenzazolium or polyazabenzazolium portion to form the dyecore structure (see U.S. Pat. No. 5,436,134, U.S. Pat. No. 5,658,751,and U.S. Pat. No. 5,863,753). Typically, desired substituents areincorporated via alkylation of the nitrogen atom of an appropriatelysubstituted quinoline, or R⁵ substituents that are aryl or heteroarylare incorporated by an Ullmann reaction of aniline or a substitutedaniline or of a pyridone or quinolone derivative (pyridone and quinoloneprecursors may also be prepared by an Ullmann reaction of theappropriately substituted precursor if the nitrogen atom ishydrogen-substituted).

Pyridone and quinolone intermediates containing a non-hydrogen group atR⁵ are particularly important precursors to a wide variety of otherpyridinium and quinolinium salts that are substituted at R⁴. Inparticular a salt is formed by treatment of the appropriate pyridone orquinolone with a strong chlorinating agent such as PCl₅, POCl₃ or SOCl₂,for instance in the reaction below. Similarly, a sulfonate can besubstituted at R⁴ by treating the pyridone or quinolone with theappropriate sulfonic acid anhydride.

The reactivity of the 2-halogenated pyridinium or quinoliniumintermediate offers a variety of synthetic methods for attachment ofvarious substituents at the 2-position, including TAIL moieties, TAILprecursors, -L-R_(x) moieties, and BRIDGE precursors. However, thereactivity of the 2-halo derivatives is preserved even after conjugationwith the benzazolium precursor, enabling conversion of the resulting dyein which R⁴ is halogen into the appropriate alkoxy, amino and thiolateanalogs, as described for the pyridinium and quinolinium precursors. Ofparticular utility for the dyes of the present invention is thedisplacement of a 2-chloro substituent by amines.

Additionally, the 2-oxo group of pyridone or quinolone precursors can bechemically reduced to derivatives in which R⁴ is H using a variety ofreagents including DIBAL-H (diisobutylaluminum hydride).

3. Th M Thine Moiety

The specific synthetic reagents used in the synthesis of the presentdyes determine the number of methine groups that connect the twoheterocyclic ring systems. When n=0, the synthesis of monomethine dyescommonly uses a combination of reagents where the methine carbon atomresults from either D on the azabenzazolium or polyazabenzazolium saltor B on the pyridinium salt being methyl and the other of D or B being areactive “leaving group” that is typically methylthio or chloro, butthat can be any leaving group that provides sufficient reactivity tocomplete the reaction, as described by Brooker et al., supra. Whether Dor B is methyl depends primarily on the relative ease of synthesis ofthe requisite precursor salts. Because 2-methyl and 4-methyl pyridinesare usually easier to prepare than their corresponding methylthioanalogs, monomethine dyes are typically prepared from precursors inwhich D=methylthio and B=methyl. The condensing reagent in the case ofmonomethine dyes is typically a weak base such as triethylamine ordiisopropylethylamine.

To synthesize trimethine dyes (n=1) both D and B are methyl. In thiscase the additional methine carbon is provided by a reagent such asdiphenylformamidine, N-methylformanilide or ethyl orthoformate. Becauseunder certain reaction conditions these same reagents can yieldsymmetrical cyanine dyes that incorporate two moles of a singlequaternary salt, it is important to use the proper synthetic conditions,and a suitable ratio of the carbon-providing reactant to the firstquaternary salt, so that the proper intermediate will be formed. Thisintermediate is treated either before or after purification with thesecond quaternary salt to form the asymmetric cyanine dye. If desired,the counterion ψ can be exchanged at this point. One can react either ofthe heteroaromatic precursor salts with the carbon-providing reagent toform the required intermediate, or form the intermediate from the morereadily available 2-methylazobenzazolium or 2-methylpolyazabenzazoliumsalts similar to the method described by Brooker et al but modified byuse of an azabenzazolium or polyazabenzazolium salt.

Synthesis of the pentamethine dyes (n=2) raises the same syntheticconcerns about controlling the formation of an asymmetric intermediate.The three-carbon fragment that is required for the additional atoms inthe methine moiety comes from a suitable precursor to malonaldehyde suchas malonaldehyde dianil, 1,1,3,3-tetramethoxypropane,1,1,3-trimethoxypropene, 3-(N-methylanilino)propenal or other reagents.The condensing agent for this reaction is usually1-anilino-3-phenylimino-1-propene (U.S. Pat. No. 2,269,234 to Sprague,1942), which generates the 2-(2-anilinovinyl)-3-methylbenzazoliumtosylate intermediate.

The introduction of desired methine substituents (R³⁰, R³¹, and/or R³²)is typically accomplished by modification of either the azabenzazoliummoiety or the quinolinium/pyridinium moiety prior to condensation of thedesired dye (Examples 28-31). Typically R³² is introduced byincorporation into the azabenzazolium moiety (Example 29), while R³⁰ isintroduced on the quinolinium/pyridinium moiety (Example 28). R³¹ may beintroduced on either heterocyclic ring system, depending on thesimplicity and accessibility of the resulting synthesis.

Those dyes having a quinoline or pyridine ring rather than a quinoliniumor pyridinium are typically prepared by either heating a azabenzazoliumor polyazabenzazolium salt that contains a good leaving group (chloride,methylthio, etc.) at the 2-position, with a 2- or 4-methyl substitutedor unsubstituted quinoline or pyridine in the presence of aceticanhydride as an activator.

Alternatively, a corresponding N-methylquinolinium cyanine dye isdealkylated (typically demethylated) using a good nucleophile, such asan alkali metal salt of a thiol (e.g. sodium thiophenoxide) in a polarsolvent (e.g. DMF, DMSO). Where R² is ethyl (or a higher alkyl), theN-methyl substituent on the pyridinium or quinolinium ring is removedselectively during dealkylation. Where the R² substituent is alsomethyl, two demethylated products may result, but the two products arereadily separated by column chromatography. Where the N-substituent is asubstituted alkyl, such as 2-methoxyethoxymethyl, other reagents, suchas a strong acid, are used to generate the corresponding pyridine orquinoline end product.

4. TAIL

As described above, TAIL is composed of three parts: LINK, SPACER andCAP. The chemical composition of SPACER is determined by the chemistryrequired to attach the heteroatom in CAP with the dye core structure viaLINK.

As described above, those dyes of the present invention that are4-substituted heterocycles wherein R⁴ is a TAIL are most convenientlysynthesized from the 2-halo substituted precursor either before or aftercondensation with the azabenzazolium or polkyazabenzazolium portion ofthe dye by a nucleophilic displacement reaction of the halogen by athiol, alkoxide, or a primary or secondary amine.

CAP may be incorporated directly into TAIL before or after condensationof the second heterocyclic ring with the azabenzazolium orpolyazabenzazolium salt, or CAP may be added or further modified at alater stage in the synthesis. For instance, when CAP is a cyclic ornon-cyclic primary, secondary or tertiary amine, CAP can be alkylated toa quaternary ammonium (Examples 10, 11). This reaction can be used toincrease the polarity of the dye and to thus restrict its penetrationthrough the membrane of living cells, and to additionally increase thedye's affinity for nucleic acids.

Precursors to TAIL include, among other functional groups, carboxylicacids, halides, alcohols and thiols. Each of these reactive groups canbe used to attach a heteroatom-containing moiety (i.e., CAP) to thedye's core structure, generally through the formation of amides, ethersor thioethers, which are incorporated into SPACER before or afterattachment of SPACER to the dye's core structure.

5. Homo- and Heterodimer Dyes

The dimeric dyes of the invention that are homodimers are typicallyprepared by condensation of two equivalents of a chemically reactivecyanine dye with one equivalent of a BRIDGE precursor, by methods knownin the art (Example 12, 13). Heterodimeric dyes are prepared similarly,but the synthesis typically requires multiple steps in order to achievethe desired final product (as described in U.S. Pat. No. 5,321,130; U.S.Pat. No. 5,436,134; U.S. Pat. No. 5,658,751; U.S. Pat. No. 5,869,689,U.S. Pat. No. 5,582,977; U.S. Pat. No. 5,401,847; U.S. Pat. No.5,565,554; and U.S. Pat. No. 5,760,201).

C. Reactive Cyanine Dyes and Dye-Conjugates

In another embodiment of the invention, the cyanine dyes of theinvention are chemically reactive, and are substituted by at least onegroup -L-R_(x), where R_(x) is the reactive group that is attached tothe dye by a covalent linkage L. R_(x) is a reactive group thatfunctions as the site of attachment for another moiety wherein thereactive group chemically reacts with an appropriate reactive orfunctional group on another substance or moiety. These reactive groupsor reactive precursor are synthesized during the formation of thepresent compounds providing present cyanine compounds that can becovalently attached to another substance, conjugated substance,facilitated by the reactive group. In this way, compounds incorporatinga reactive group (R_(x)) can be covalently attached to a wide variety ofbiomolecules or non-biomolecules that contain or are modified to containfunctional groups with suitable reactivity, resulting in chemicalattachment of the conjugated substance (S_(C)), represented by -L-S_(C).This conjugation typically confers the nucleic acid- and/or poly(aminoacid)-sensing abilities of the cyanine dye on the conjugated substance.However, the present dye compounds can also function as reportermolecules for the conjugated substance wherein the sensing ability ofthe compounds is not employed. The reactive group and functional groupare typically an electrophile and a nucleophile that can generate acovalent linkage. Alternatively, the reactive group is aphotoactivatable group, and becomes chemically reactive only afterillumination with light of an appropriate wavelength. Typically, theconjugation reaction between the reactive group and the substance to beconjugated results in one or more atoms of the reactive group R_(x) tobe incorporated into a new linkage attaching the compound of theinvention to the conjugated substance Sc. Selected examples offunctional groups and linkages are shown in Table 3, where the reactionof an electrophilic group and a nucleophilic group yields a covalentlinkage.

TABLE 3 Examples of some routes to useful covalent linkages withelectrophile and nucleophile reactive groups Electrophilic GroupNucleophilic Group Resulting Covalent Linkage activated esters*amines/anilines carboxamides acyl azides** amines/anilines carboxamidesacyl halides amines/anilines carboxamides acyl halides alcohols/phenolsesters acyl nitriles alcohols/phenols esters acyl nitrilesamines/anilines carboxamides aldehydes amines/anilines imines aldehydesor ketones hydrazines hydrazones aldehydes or ketones hydroxylaminesoximes alkyl halides amines/anilines alkyl amines alkyl halidescarboxylic acids esters alkyl halides thiols thioethers alkyl halidesalcohols/phenols ethers alkyl sulfonates thiols thioethers alkylsulfonates carboxylic acids esters alkyl sulfonates alcohols/phenolsethers anhydrides alcohols/phenols esters anhydrides amines/anilinescarboxamides aryl halides thiols thiophenols aryl halides mines rylamines aziridines thiols thioethers boronates glycols boronate esterscarboxylic acids amines/anilines carboxamides carboxylic acids alcoholsesters carboxylic acids hydrazines hydrazides carbodiimides carboxylicacids N-acylureas or anhydrides diazoalkanes carboxylic acids estersepoxides thiols thioethers haloacetamides thiols thioethershalotriazines amines/anilines aminotriazines halotriazinesalcohols/phenols triazinyl ethers imido esters amines/anilines amidinesisocyanates amines/anilines ureas isocyanates alcohols/phenols urethanesisothiocyanates amines/anilines thioureas maleimides thiols thioethersphosphoramidites alcohols phosphite esters silyl halides alcohols silylethers sulfonate esters amines/anilines alkyl amines sulfonate estersthiols thioethers sulfonate esters carboxylic acids esters sulfonateesters alcohols ethers sulfonyl halides amines/anilines sulfonamidessulfonyl halides phenols/alcohols sulfonate esters *Activated esters, asunderstood in the art, generally have the formula —COΩ, where Ω is agood leaving group (e.g. oxysuccinimidyl (—OC₄H₄O₂) oxysulfosuccinimidyl(—OC₄H₃O₂—SO₃H), -1-oxybenzotriazolyl (—OC₆H₄N₃); or an aryloxy group oraryloxy substituted one or more times by electron withdrawingsubstituents such as nitro, fluoro, chloro, cyano, or trifluoromethyl,or combinations thereof, used to form activated aryl esters; or acarboxylic acid activated by a carbodiimide to form an anhydride ormixed anhydride —OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a) and R^(b),which may be the same or different, are C₁-C₆ alkyl, C₁-C₆perfluoroalkyl, or C₁-C₆ alkoxy; or cyclohexyl, 3-dimethylaminopropyl,or N-morpholinoethyl). **Acyl azides can also rearrange to isocyanates

The cyanine compounds of the present invention typically comprise alinker when the dye compound is substituted with a reactive group or aconjugated substance wherein the linker is used to covalently attach aconjugated substance or reactive group to the compound. When present,the linker is a single covalent bond or a series of stable bonds. Thus,the conjugated substance or reactive group may be directly attached(where Linker is a single bond) to the cyanine compounds or attachedthrough a series of stable bonds. When the linker is a series of stablecovalent bonds the linker typically incorporates 1-20 nonhydrogen atomsselected from the group consisting of C, N, O, S and P. In addition, thecovalent linkage can incorporate a platinum atom, such as described inU.S. Pat. No. 5,714,327. When the linker is not a single covalent bond,the linker may be any combination of stable chemical bonds, optionallyincluding, single, double, triple or aromatic carbon-carbon bonds, aswell as carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygenbonds, sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygenbonds, phosphorus-nitrogen bonds, and nitrogen-platinum bonds. Typicallythe linker incorporates less than 15 nonhydrogen atoms and are composedof any combination of ether, thioether, thiourea, amine, ester,carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromaticbonds. Typically the linker is a single covalent bond or a combinationof single carbon-carbon bonds and carboxamide, sulfonamide or thioetherbonds. The bonds of the linker typically result in the followingmoieties that can be found in the linker: ether, thioether, carboxamide,thiourea, sulfonamide, urea, urethane, hydrazine, alkyl, aryl,heteroaryl, alkoky, cycloalkyl and amine moieties. The longest linearsegment of the linkage L preferably contains 4-10 nonhydrogen atomsincluding one or two heteroatoms. Examples of L include substituted orunsubstituted polymethylene, arylene, alkylarylene or arylenealkyl. Inone embodiment, L contains 1-6 carbon atoms; in another, L is athioether linkage. In yet another embodiment, L has the formula—(CH₂)_(a)(CONH(CH₂)_(b))_(z)—, where a is an integer from 0-5, b is aninteger from 1-5 and z is 0 or 1.

Any combination of linkers may be used to attach the Rx or Sc and thecyanine compounds together, typically a compound of the presentinvention when attached to more than one Rx or Sc will have one or twolinkers attached that may be the same or different, The linker may alsobe substituted to alter the physical properties of the carbocyaninecompounds, such as solubility and spectral properties of the compound.

The -L-Rx moiety, or precursor to the -L-Rx moiety, is synthesized onthe appropriate precursor during the synthesis of the cyanine dyecompound. The, -L-R_(x) moieties are typically prepared from 2-halosubstituted heterocyclic precursors, either before or after condensationwith the azabenzazolium or polyazabenzazolium portion of the dye by anucleophilic displacement reaction of the halogen by a thiol, alkoxide,or a primary or secondary amine, which is then incorporated into thecovalent linkage L.

The -L-R_(x) group can be bound to the dye at R², R³, R⁴, R⁵, R⁶, R⁷,R¹¹, R¹², R¹³, or R¹⁴. Alternatively, the -L-R_(x) moiety is bound toone of the aromatic carbons of the aza-benzazole ring system. Typically,-L-R_(x) is bound to the dye at R², R⁴ or R⁵. Preferably -L-R_(x) isbound to the dye at R⁴ or R⁵.

The selection of covalent linkage to attach the dye to the conjugatedsubstance typically depends on the chemically reactive functional groupon the substance to be conjugated. The types of functional groupstypically present on the organic or inorganic substances include, butare not limited to, amines, thiols, alcohols, phenols, aldehydes,ketones, phosphates, imidazoles, hydrazines, hydroxylamines,disubstituted amines, halides, epoxides, sulfonate esters, purines,pyrimidines, carboxylic acids, or a combination of these groups. Asingle type of reactive site may be available on the substance (typicalfor polysaccharides), or a variety of sites may occur (e.g. amines,thiols, alcohols, phenols), as is typical for proteins. A conjugatedsubstance may be conjugated to more than one dye, which may be the sameor different, or to a substance that is additionally modified by ahapten. Although some selectivity can be obtained by careful control ofthe reaction conditions, selectivity of labeling is best obtained byselection of an appropriate reactive dye.

Typically, R_(x) will react with an amine, a thiol or an alcohol. In oneembodiment, R_(x) is an acrylamide, an activated ester of a carboxylicacid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, anamine, an anhydride, an aniline, an aryl halide, an azide, an aziridine,a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, ahalotriazine, a hydrazine, an imido ester, an isocyanate, anisothiocyanate, a maleimide, a phosphoramidite, a sulfonyl halide, or athiol group. Preferably, R_(x) is a carboxylic acid, a succinimidylester, an amine, a haloacetamide, an alkyl halide, a sulfonyl halide, anisothiocyanate, a maleimide group or an azidoperfluorobenzamido group.

Prior to use in preparation of conjugates to form -L-Sc moieties,chemically reactive forms of the dyes of the invention are typicallydissolved in water or a water-miscible such as a lower alcohol,dimethylformamide (DMF), dimethylsuifoxide (DMSO), acetone,acetonitrile, tetrahydrofuran (THF), dioxane or acetonitrile. Thepreparation of dye conjugates using reactive dyes is well documented,Haugland, MOLECULAR PROBES, INC. HANDBOOK OF FLUORESCENT PROBES ANDRESEARCH CHEMICALS, 1996 and Brinkley, BIOCONJUGATE CHEM., 3, 2 (1992).Conjugates typically result from mixing appropriate reactive cyaninedyes and the substance to be conjugated. In a suitable solvent in whichboth are soluble, using methods well known in the art, followed byseparation of the conjugate from any unreacted dye and by-products.These dyes are typically combined with the sample under conditions ofconcentration, stoichiometry, pH, temperature and other factors thataffect chemical reactions that are determined by both the reactivefunctional groups on the dye and the expected site of modification onthe molecule to be modified. These factors are generally well known inthe art of forming bioconjugates (Haugland et al. “Coupling ofAntibodies with Biotin” THE PROTEIN PROTOCOLS HANDBOOK, J. M. Walker,ed., Humana Press, (1996); Haugland “Coupling of Monoclonal Antibodieswith Fluorophores” METHODS IN MOLECULAR BIOLOGY, VOL. 45: MONOCLONALANTIBODY PROTOCOLS, W. C. Davis, Ed. (1995)). For those reactive dyesthat are photoactivated, conjugation requires illumination of thereaction mixture to activate the reactive dye. The dye-conjugate is usedin solution or lyophilized and stored for later use.

A variety of dye-conjugates may be prepared using the reactive dyes ofthe invention, including present dye conjugates of antigens, steroids,vitamins, drugs, haptens, metabolites, toxins, environmental pollutants,amino acids, peptides, proteins, nucleic acids, nucleic acid polymers,carbohydrates, lipids, and polymers. In another embodiment, theconjugated substance is an amino acid, peptide, protein, polysaccharide,nucleotide, oligonucleotide, nucleic acid, hapten, drug, lipid,phospholipid, lipoprotein, lipopolysaccharide, liposome, lipophilicpolymer, polymer, polymeric microparticle, biological cell or virus. Inone aspect of the invention, the conjugated substance is labeled with aplurality of dyes of the present invention, which may be the same ordifferent.

Typically, the conjugated substance (S_(C)) is an amino acid, a peptide,a protein, a polysaccharide, a nucleotide, an oligonucleotide, a nucleicacid, a lipid, a polymeric microparticle, a biological cell, or a virus.The most preferred conjugated substances are conjugates of haptens,nucleotides, oligonucleotides, nucleic acid polymers, proteins, orpolysaccharides. Most preferably, the conjugated substance is a nucleicacid, or a substance that interacts in a specific fashion with nucleicacids, such as DNA-binding proteins.

In one embodiment, the conjugated substance (S_(c)) is an amino acid(including those that are protected or are substituted by phosphates,carbohydrates, or C₁ to C₂₂ carboxylic acids), or is a polymer of aminoacids such as a peptide or protein. Preferred conjugates of peptidescontain at least five amino acids, more preferably 5 to 36 amino acids.Preferred peptides include, but are not limited to, neuropeptides,cytokines, toxins, protease substrates, and protein kinase substrates.Also preferred are peptides that serve as organelle localizationpeptides, that is, peptides that serve to target the conjugated dye forlocalization within a particular cellular substructure by cellulartransport mechanisms. Preferred protein conjugates include enzymes,antibodies, tectins, glycoproteins, histones, albumins, lipoproteins,avidin, streptavidin, protein A, protein G, phycobiliproteins and otherfluorescent proteins, hormones, toxins and growth factors. Typically,the conjugated protein is an antibody, an antibody fragment, avidin,streptavidin, a toxin, a lectin, or a growth factor. Preferred haptensinclude biotin, digoxigenin and fluorophores.

In another embodiment, the conjugated substance (S_(c)) is a nucleicacid base, nucleoside, nucleotide or a nucleic acid polymer, optionallycontaining an additional linker or spacer for attachment of afluorophore or other ligand, such as an alkynyl linkage (U.S. Pat. No.5,047,519), an aminoallyl linkage (U.S. Pat. No. 4,711,955) or otherlinkage. Preferably, the conjugated nucleotide is a nucleosidetriphosphate or a deoxynucleoside triphosphate or a dideoxynucleosidetriphosphate.

Preferred nucleic acid polymer conjugates are single- or multi-stranded,natural or synthetic DNA or RNA oligonucleotides, or DNA/RNA hybrids, orincorporating an unusual linker such as morpholine derivatizedphosphates (AntiVirals, Inc., Corvallis Oreg.), or peptide nucleic acidssuch as N-(2-aminoethyl)glycine units, where the nucleic acid containsfewer than 50 nucleotides, more typically fewer than 25 nucleotides. Thedye is optionally attached via one or more purine or pyrimidine basesthrough an amide, ester, ether or thioether bond; or is attached to thephosphate or carbohydrate by a bond that is an ester, thioester, amide,ether or thioether. Alternatively, the dye is attached by formation of anon-covalent association of the nucleic acid and a photoreactive dye ofthe invention, followed by illumination, resulting in covalently bounddye. Nucleotide conjugates of the invention can be incorporated by someDNA polymerases and can be used for in situ hybridization and nucleicacid sequencing (e.g., U.S. Pat. Nos. 5,332,666; 5,171,534; and4,997,928, all incorporated by reference; and WO Appl. 94/05688).

In another embodiment, the conjugated substance (S_(c)) is acarbohydrate or polyol that is typically a polysaccharide, such asdextran, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, starch,agarose and cellulose, or is a polymer such as a poly(ethylene glycol).Preferred polysaccharide conjugates are dextran or FICOLL conjugates.

In another embodiment, the conjugated substance (S_(c)), is a lipid(typically having 6-25 carbons), including glycolipids, phospholipids,and sphingolipids. Alternatively, the conjugated substance is a lipidvesicle, such as a liposome, or is a lipoprotein (see below). Somelipophilic substituents are useful for facilitating transport of theconjugated dye into cells or cellular organelles.

Furthermore, the conjugates are optionally dye-conjugates of polymers,polymeric particles, polymeric microparticles including magnetic andnon-magnetic microspheres, polymeric membranes, conducting andnon-conducting metals and non-metals, and glass and plastic surfaces andparticles. Conjugates are typically prepared by chemical modification ofa polymer that contains functional groups with suitable chemicalreactivity. The conjugated polymer may be organic or inorganic, naturalor synthetic. In a preferred embodiment, the dye is conjugated to apolymer matrix, such as a polymeric particle or membrane, includingmembranes suitable for blot assays for nucleic acids or proteins. Inanother embodiment, the conjugated substance is a glass or silica, whichmay be formed into an optical fiber or other structure. In anotherembodiment, the conjugated polymer is poly(ethylene glycol), apoly(acrylate) or a poly(acrylamide).

In one embodiment, the conjugated substance is a specific binding pairmembers wherein the dye is conjugated to a specific binding pair memberand used to detect nucleic acids or poly(amino acids). Alternatively,the presence of the labeled specific binding pair member indicates thelocation of the complementary member of that specific binding pair; eachspecific binding pair member having an area on the surface or in acavity which specifically binds to, and is complementary with, aparticular spatial and polar organization of the other. In thisinstance, the dye compounds of the present invention function as areporter molecule for the specific binding pair and not a dye selectivefor poly(amino acids) or nucleic acids.

TABLE 4 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor folate folate binding protein toxin toxin receptorcarbohydrate lectin or carbohydrate receptor peptide peptide receptorprotein protein receptor enzyme substrate enzyme DNA (RNA) cDNA (cRNA)†hormone hormone receptor ion chelator *IgG is an immunoglobulin †cDNAand cRNA are the complementary strands used for hybridizationD. Methods of Use

The use of the invention comprises combining a dye (monomer or dimer) ordye-conjugate of the present invention with a sample that contains or isthought to contain the desired analyte, incubating the mixture of dyeand sample for a time sufficient for the dye to combine with the analytein the sample and to form one or more dye-analyte complexes having adetectable fluorescent signal. The characteristics of the dye-analytecomplex. Including the presence, location, intensity, excitation andemission spectra, fluorescence polarization, fluorescence lifetime,photobleaching rate and other physical properties of the fluorescentsignal can be used to detect, differentiate, sort, quantitate, and/oranalyze aspects or portions of the sample. The dyes of the invention areoptionally used in conjunction with one or more additional detectionreagents (preferably detectably different fluorescent reagents),including dyes of the same class having different spectral properties.

Therefore, a method for determining the presence of an analyte ofinterest in a sample, comprises the steps of:

-   -   a) combining said sample with a staining solution, wherein said        staining solution comprises one or more dyes of the present        invention;    -   b) incubating said sample and said staining solution for a        sufficient amount of time to form a dye-analyte complex;    -   c) illuminating said complex with an appropriate wavelength        whereby the presence of said analyte is determined.

Typically the analyte of interest is a nucleic acid polymer or alipid-complexed poly(amino acid). However when a dye-conjugate ispresent in the staining solution the analyte may be any compound theconjugated substance has an affinity for. Thus, in one embodiment of theinvention, the analyte is a nucleic acid polymer, such as DNA or RNA. Inanother embodiment of the invention, the analyte is a lipid-complexedpoly(amino acid). By poly(amino acid) is meant any polymer of amino acidsubunits, artificial or natural, such as peptides and proteins, andincluding glycoproteins, lipoproteins and other modified proteins.

The staining solution is preferably an aqueous or aqueous misciblesolution that is compatible with the sample and the intended use. Forbiological samples, where minimal perturbation of cell morphology orphysiology is desired, the staining solution is selected accordingly.For solution assays, the staining solution preferably does not perturbthe native conformation of the analyte undergoing evaluation.

The dyes have greater stability in buffered solutions than in wateralone; and agents that reduce the levels of free oxygen radicals, suchas β-mercaptoethanol, contribute to the stability of the dyes. Whenstaining nucleic acids, high concentrations of organic solvents,cations, and oxidizing agents generally reduce fluorescence of thedye-nucleic acid complex, as does the ionic detergent sodium dodecylsulfate (SDS) at concentrations >0.01%. A number of staining solutionadditives, however, do not interfere with the fluorescence of thedye-nucleic acid complex (e.g. urea up to 8 M; CsCl up to 1 g/mL;formamide up to 50% of the solution; and sucrose up to 40%). Nonionicdetergents such as Tween 20, NP40 or Triton X-100 at concentration ofless than or equal to 0.1%, DMSO at concentrations less than or equal to10%, and ethanol at concentrations of less than or equal to about 10%stabilize the dye-buffer staining solution, but can interfere withstaining of poly(amino acids).

Although typically used in an aqueous or aqueous miscible solution, thestaining solution is typically prepared by first dissolving the dye in awater-miscible organic solvent such as dimethylsulfoxide (DMSO),dimethylformamide (DMF), or a lower alcohol, such as methanol orethanol. This stock solution is typically prepared at a concentration ofgreater than about 100-times that used in the final staining solution,then diluted one or more times with an aqueous solvent or a buffersolution such that the dye is present in an effective amount. The buffersolution may be, for example, a buffered saline (preferablynon-phosphate for some viability discrimination applications), or aTris(hydroxymethyl)aminomethane (TRIS) buffer (preferably containingEDTA). Typically, the dye is first dissolved in 100% DMSO, and thendiluted with buffer.

The staining solution, containing an effective amount of dye, is thencombined with a sample for a sufficient amount of time to form adye-analyte complex. An effective amount of dye is the amount of dyesufficient to give a detectable fluorescence response in combinationwith the desired analyte. The dye concentration in the staining solutionmust be sufficient both to contact the analyte in the sample and tocombine with the analyte in an amount sufficient to give a signal, buttoo much dye may cause problems with background fluorescence. Theoptimal concentration and composition of the staining solution isdetermined by the nature of the sample (including physical, biological,biochemical and physiological properties), the nature of the dye-analyteinteraction (including the transport rate of the dye to the site of theanalyte), and the nature of the analysis being performed, and can bedetermined using standard procedures, similar to those described inexamples below.

The analyte of interest is optionally enclosed within a biologicalstructure (i.e. an organism or a discrete unit of an organism), free insolution (including solutions that contain biological structures),immobilized in or on a solid or semi-solid material, or is extractedfrom a biological structure (e.g. from lysed cells, tissues, organismsor organelles). Preferably, the analyte is present in an aqueousenvironment in order to facilitate contact with the dye.

The sample is combined with the staining solution by any means thatfacilitates contact between the dye and the analyte. Typically thecontact occurs through simple mixing, as in the case where the sample isa solution. A staining solution containing the dye may be added to theanalyte solution directly or may contact the analyte solution in aliquid separation medium such as an electrophoretic liquid, sievingmatrix or running buffer, or in a sedimentation (e.g. sucrose) orbuoyant density gradient (e.g. containing CsCl), or on an inert matrix,such as a blot or gel, a testing strip, or any other solid or semi-solidsupport. Suitable supports also include, but are not limited to,polymeric microparticles (including paramagnetic microparticles),polyacrylamide and agarose gels, nitrocellulose filters, computer chips(such as silicon chips), natural and synthetic membranes, liposomes andalginate hydrogels, and glass (including optical filters), and othersilica-based and plastic support. The dye is optionally combined withthe analyte solution prior to undergoing gel or capillaryelectrophoresis, gradient centrifugation, or other separation step,during separation, or after the nucleic acids or lipid-complexedproteins undergo separation. Alternatively, the dye is combined with aninert matrix or solution in a capillary prior to addition of the analytesolution, as in pre-cast gels, capillary electrophoresis or preformeddensity or sedimentation gradients.

The sample is combined with the dye for a time sufficient to form thefluorescent dye-analyte complex, preferably the minimum time required togive a high signal-to-background ratio. Although all of the present dyesare nucleic acid stains and lipid-complexed protein stains, detectablefluorescence within biological structures or in gels requires entry ofthe dye across the biological membrane or into gels. Optimal stainingwith a particular dye is dependent upon the physical and chemical natureof the individual sample and the sample medium, as well as the propertybeing assessed. The optimal time is usually the minimum time requiredfor the dye, in the concentration being used, to achieve the highesttarget-specific signal while avoiding degradation of the sample overtime and minimizing all other fluorescent signals due to the dye. Forexample, where the dye is chosen to be selective for a particularnucleic acid polymer or type of cell, the optimal time is usually theminimum time required to achieve the highest signal on that polymer ortype of cell, with little to no signal from other nucleic acids or othercell types. Over time, undesirable staining may occur as even very lowrates of diffusion may transport small amounts of the very sensitivedyes into other cell types, or staining selectivity may be lost as thecell membranes degrade, or as nucleases degrade nucleic acid polymers incell-free systems.

Preferably, the dye is combined with the sample at a temperature optimalfor biological activity of the analyte within the operating parametersof the dyes (usually between 5° C. and 50° C., with reduced stability ofthe dyes at higher temperatures). For in vitro assays, the dye istypically combined with the sample at about room temperature (23° C.).At room temperature, detectable fluorescence in a solution of nucleicacids is essentially instantaneous depending on the sensitivity of theinstrumentation that is used; fluorescence in solutions is generallyvisible by eye within 5 seconds after the dye is added, and is generallymeasurable within 2 to 5 minutes, although reaching equilibrium stainingmay take longer. In particular, staining of lipid-complexed proteins insolution may be slower than equivalent staining of nucleic acids. Wherea biological process is underway during in vitro analysis (e.g. in vitrotranscription, replication, splicing, or recombination), the rapidlabeling that occurs with the subject dyes avoids perturbation ofbiological system that is being observed. Gel staining at roomtemperature usually takes from 5 minutes to 2 hours depending on thethickness of the gel and the percentage of agarose or polyacrylamide, aswell as the degree of cross-linking. Typically, post-stained minigelsstain to equilibrium in 20-45 minutes. For cells and other biologicalstructures, transport of dyes across membranes is required whether themembranes are intact or disrupted. For preferred embodiments, visiblydetectable fluorescence is obtained at room temperature within 15-20minutes of incubation with cells, commonly within about 5 minutes. Someembodiments give detectable fluorescence inside cells in less than about2 minutes. This property is useful for observing nuclear structure andrearrangement, for example such as occurs during mitosis or apoptosis.Some of the dyes are generally not permeant to live cells with intactmembranes; other dyes are generally permeant to eukaryotes but not toprokaryotes; still other dyes are only permeant to cells in which thecell membrane integrity has been disrupted (e.g. some dead cells). Therelative permeability of the cell membrane to the dyes is determinedempirically, e.g. by comparison with staining profiles or stainingpatterns of killed cells. The dye with the desired degree ofpermeability, and a high absorbance and quantum yield when bound tonucleic acids or lipid-complexed proteins, is selected to be combinedwith the sample.

The presence, location, and distribution of the analyte is detectedusing the spectral properties of the fluorescent dye-analyte complex.Spectral properties means any parameter that may be used to characterizethe excitation or emission of the dye-nucleic acid complex includingabsorption and emission wavelengths, fluorescence polarization,fluorescence lifetime, fluorescence intensity, quantum yield, andfluorescence enhancement. Typically the spectral properties ofexcitation and emission wavelength are used to detect the dye complexes.The wavelengths of the excitation and emission bands of the dyes varywith dye composition to encompass a wide range of illumination anddetection bands. This allows the selection of individual dyes for usewith a specific excitation source or detection filter. In particular,complexes formed with dyes having a monomethine moiety (n=0) generallymatch their primary excitation band with the 532 nm excitation line ofthe frequency-doubled Nd-Yag laser, the 543 nm excitation line of thegreen He—Ne laser, the strong 546 nm emission of the Hg-arc lamp andsometimes the 568 nm excitation line of the Kr laser; whereas those withdyes with a trimethine moiety (n=1) primarily tend to match longwavelength excitation sources such as the orange HeNe laser (594 nm),the red HeNe laser (633 nm), the Kr laser (568 or 647 nm) and certainlaser diodes, in particular the 635 nm laser diode; and complexes formedwith dyes having a pentamethine moiety (n=2) primarily match very longexcitation sources such as laser diodes or light emitting diodes (LEDs).In addition to the primary excitation peak in the visible range, thedye-nucleic acid complexes and dye-lipid-complexed protein complexes ofthe invention have a secondary absorption peak that permits excitationwith UV illumination. Dyes with n=1 and n=2 typically form complexesthat permit excitation beyond 600 nm.

Typically, the sample is excited by a light source capable of producinglight at or near the wavelength of maximum absorption of the fluorescentanalyte-complex, such as an ultraviolet or visible wavelength emissionlamp, an arc lamp, a laser, or even sunlight or ordinary roomlight.Preferably the sample is excited with a wavelength within 20 nm of themaximum absorption of the fluorescent complex. Although excitation by asource more appropriate to the maximum absorption band of thedye-analyte complex results in higher sensitivity, the equipmentcommonly available for excitation of samples can be used to excite thedyes of the present invention.

The fluorescence of the complex is detected qualitatively orquantitatively by detection of the resultant light emission at awavelength of greater than about 450 nm, preferably greater than about480 nm, more preferably at greater than about 500 nm. The emission isdetected by means that include visual inspection, CCD cameras, videocameras, photographic film, or the use of instrumentation such as laserscanning devices, fluorometers, photodiodes, quantum counters, platereaders, epifluorescence microscopes, scanning microscopes, confocalmicroscopes, flow cytometers, capillary electrophoresis detectors, or bymeans for amplifying the signal such as a photomultiplier tube. Manysuch instruments are capable of utilizing the fluorescent signal to sortand quantitate cells or quantitate the nucleic acids. Dyes can beselected to have emission bands that match commercially available filtersets such as that for fluorescein, tetramethylrhodamine or for detectingmultiple fluorophores with several excitation and emission bands.

The source and type of sample, as well as the use of the dye, willdetermine which dye characteristics, and thus which dyes, will be mostuseful for staining a particular sample. For most applications, dyes areselected to give a quantum yield greater than about 0.1, more usefully0.2 or 0.3, preferably greater than 0.5, when associated with ananalyte; preferably the dyes have a quantum yield <0.02 and morepreferably <0.01 or <0.005 when present in solution and not associatedwith an analyte. Preferably, upon associating with an analyte, the dyeof the invention will exhibit a fluorescence enhancement greater thanabout 50 fold, preferably greater than 100 fold, more preferably greaterthan 200 fold and most preferably greater than 500 fold. Where thefluorescence of the dye-analyte complex or dye conjugate is detectedutilizing sustained high intensity illumination (e.g. microscopy), dyeswith a rate of photobleaching lower than commonly used dyes (e.g.fluorescein) are preferred, particularly for use in live cells. Wherethe dye must penetrate cell membranes or a gel, more permeant dyes arepreferred, although some cells readily take up dyes that are shown to beimpermeant to other cells by means other than passive diffusion acrosscell-membranes, e.g. by phagocytosis or other types of ingestion. Dyesthat rapidly and readily penetrate cells do not necessarily rapidlypenetrate gels. In applications where the nucleic acids are stained on agel, the dye is also selected to have a relatively high binding affinity(preferably K_(d)<10⁻⁶ M); whereas in applications where the nucleicacid will be pre-stained prior to undergoing a separation step, such asgel or capillary electrophoresis, even higher binding affinity(preferably K_(d)<10⁻⁸ M) is preferred to ensure good separation. Instaining nucleic acids in solution, high binding affinity translatesinto greater sensitivity to small amounts of nucleic acid, but dyes witha moderate binding affinity (preferably 10⁻⁶ M<K_(d)<10⁻⁸ M) are moreeffective over a greater dynamic range. The photostability, bindingaffinity, quantum yield, and fluorescence enhancement of dyes aredetermined according to standard methods known in the art.

In a preferred embodiment, the dyes of the present invention areemployed to detect the presence of lipid-poly(amino acids). In thisinstance the poly(amlno acids) are typically immobilized on a solid orsemi-solid matrix, but alternatively may be present in solution providedthe correct concentration of lipid is present. Therefore, a method ofthe present invention for detecting lipid complexed poly(amino acids) ina sample comprises:

-   -   a) combining said sample with a staining solution, wherein said        staining solution comprises one or more dyes of the present        invention;    -   b) incubating said sample and said staining solution for a        sufficient amount of time to form a dye-analyte complex;    -   c) illuminating said complex with an appropriate wavelength        whereby the presence of said analyte is determined.

The sample containing a poly(amino acid) is in a solution, orimmobilized on a solid or semi-solid matrix such as a blot orelectrophoretic gel, the dye is preferably combined with the sample inan effective amount in the presence of a low percentage of a lipid. Theamount of lipid or detergent required is typically a concentrationavailable from a solution of 0.05-2.0% sodium dodecyl sulfate (SDS).Although the dyes of the invention may stain some proteins in theabsence of lipid complexation, both the association of the dye with theprotein and the resulting fluorescent signal are enhanced by thepresence of a lipid complexing agent. Preferably the lipid complexingagent is an amphiphilic lipid, such as a detergent. Due to thelimitation of a detergent for staining all proteins, the lipid-complexedprotein is typically not associated with a biological cell but is freein solution or is immobilized on a solid or semi-solid matrix or is inan electrophoretic matrix or chamber, such as one used for gel orcapillary electrophoresis.

The detergent is optionally added simultaneously with or as part of thesample or the staining solution, or is added thereafter to the combinedmixture. The detergent is any amphiphilic surface active agent orsurfactant that serves to coat the poly(amino acids) (i.e.non-covalently associate with the poly(amino acid)), or is a mixture ofmore than one detergent. Useful detergents include non-ionic, cationic,anionic, amphoteric and fluorinated surfactants. While there are avariety of detergents that are commercially available, includingnon-ionic, cationic, and anionic detergents, any detergent that isutilized in protein gel electrophoresis is a preferred detergent for thepresent invention. Typically, the detergent is an anionic detergent,preferably an alkyl sulfate or alkyl sulfonate salt. More preferably,the detergent is sodium dodecyl sulfate (SDS), sodium octadecyl sulfate,or sodium decyl sulfate, or a mixture thereof. Most preferably, thedetergent is sodium dodecyl sulfate.

The dyes of the invention typically stain micelles, when present, so itis preferred that any detergent or detergent mixture present in thesample mixture, staining mixture, or combined mixture be present belowthe critical micelle concentration (CMC) for that detergent.

Combination of the dye with lipid-complexed proteins is facilitated whenthe proteins are first heated in the presence of detergent, including totemperatures in excess of 90° C. This heating step optionally occurs inthe presence of the dye of the invention, although exposure to elevatedtemperatures may effect the stability of the dye.

The poly(amino acids) are optionally a synthetic or naturally occurringpoly(amino acid), such as a peptide, polypeptide or protein. Poly(aminoacids) that are stained and analyzed according to the present methodoptionally incorporate non-peptide regions (covalently ornon-covalently) including lipid (lipopeptides and lipoproteins),phosphate (phosphopeptides and phosphoproteins), and/or carbohydrate(glycopeptides and glycoproteins) regions; or incorporate metal chelatesor other prosthetic groups or non-standard side chains; or aremulti-subunit complexes, or incorporate other organic or biologicalsubstances, such as nucleic acids. The poly(amino acids) are optionallyrelatively homogeneous or heterogeneous mixtures of poly(amino acids).In one aspect of the invention the poly(amino acids) are enzymes,antibodies, transcription factors, secreted proteins, structuralproteins, or binding factors, or combinations thereof. The poly(aminoacids) in the sample mixture are optionally covalently or non-covalentlybound to a solid surface, such as a glass slide, multi-well plate,microtiter plate well, plastic pin or bead, or semiconductor material,or they are unbound. The staining of a poly(amino acid) that is bound toan analyte on a solid surface indicates the presence of the analyte aswell as that of the poly(amino acid).

The poly(amino acids) are optionally unmodified, or have been treatedwith a reagent so as to enhance or decrease the mobility of thepoly(amino acid) in an electrophoretic gel. Such reagents may modifypoly(amino acids) by complexing with the peptide (to decreasemigration), by cleaving selected peptide bonds (to increase migration ofthe resulting fragments), by changing the relative charge on the protein(as by phosphorylation or dephosphorylation) or by covalent coupling ofa constituent such as occurs during glycosylation. The presence orinteraction of such a reagent in the sample mixture is detected by thechange in electrophoretic mobility of the treated poly(amino acids),relative to untreated poly(amino acids) having the same originalcomposition, so that the distribution of the dye-poly(amino acid)complex indicates the presence of another analyte.

Typically the poly(amino acids) in the sample mixture have a molecularweight greater than about 500 daltons. More typically the poly(aminoacids) are more than 800 daltons. Smaller polymers of amino acids (inthe <1000 dalton range) are generally difficult to separate from thedetergent front on denaturing gels, and typically do not adhere tofilter membranes, but are still readily detected in solution. There isno precise upper limit on the size of the poly(amino acids) that may bestained and detected, except that they can not be so bulky that theyprecipitate out of solution, which also depends in part on the relativehydrophobicity of the poly(amino acid). Furthermore, poly(amino acids)greater than about 200,000 daltons are generally not effectivelyresolved with current gel technology. The poly(amino acids) presentoptionally have essentially the same molecular weight or fall within arange of molecular weights. In one embodiment of the invention, thepoly(amino acids) present are a mixture of poly(amino acids) ofdifferent molecular weights that are used as molecular weight standards.A typical such mixture contains equal mass quantities of myosin,β-galactosidase, phosphorylase B, bovine serum albumin, ovalbumin,carbonic anhydrase, trypsin inhibitor, lysozyme and aprotinin.

Where the sample mixture is an aqueous solution, the poly(amino acids)of the sample mixture are typically present in a concentration of 10ng/mL-50 μg/mL, more preferably in a concentration of 30 ng/mL-10 μg/mL,most preferably in a concentration of 50 ng/mL-5 μg/mL. Where the samplemixture is an electrophoretic gel, the poly(amino acids) of the samplemixture are typically present in a concentration of 1 ng/band-4 μg/band.

The poly(amino acids) are obtained from a variety of sources; suchsources include biological fermentation media and automated proteinsynthesizers, as well as prokaryotic cells, eukaryotic cells, virusparticles, tissues, and biological fluids. Suitable biological fluidsinclude, but are not limited to, urine, cerebrospinal fluid, blood,lymph fluids, interstitial fluid, cell extracts, mucus, saliva, sputum,stool, physiological or cell secretions or other similar fluids.

Depending on the source of the sample mixture, it optionally containsdiscrete biological ingredients other than the desired poly(aminoacids), including poly(amino acids) other than those desired, aminoacids, nucleic acids, carbohydrates, and lipids, which may or may not beremoved in the course of, prior to, or after staining. In one aspect ofthe invention, the poly(amino acids) in the sample mixture are separatedfrom each other or from other ingredients in the sample by mobility(e.g. electrophoretic gel or capillary) or by size (e.g. centrifugation,pelleting or density gradient), or by binding affinity (e.g. to a filtermembrane) in the course of the method. In another aspect of theinvention, the sample mixture thought to contain the poly(amino acids)has undergone separation. In yet another aspect of the invention, thepoly(amino acids) are not separated. Although lipid assemblies such asintact or fragmented biological membranes (e.g. membranes of cells andorganelles), liposomes, or detergent micelles, and other lipids areoptionally present in the sample mixture; the presence of large amountsof lipids, particularly lipid assemblies, Increases background labelingdue to non-specific staining. For effective detection of labeledpoly(amino acids), intact or fragmented biological membranes in thesample mixture are preferably removed, destroyed or dispersed prior toor in the course of labeling with this method. Typically treatment ofthe sample mixture by standard methods to remove some or all of suchlipids, such as ammonium sulfate precipitation, solvent extraction ortrichloroacetic acid precipitation is used. Alternatively oradditionally, lipids are removed in the course of labeling thepoly(amino acids) such as by electrophoretic separation or otherseparation techniques (e.g. centrifugation, including gradients), or aredisrupted or dispersed below the concentration at which they assembleinto micelles (critical micelle concentration) by mechanical means suchas sonication. Naturally occurring lipids that are present below theircritical micelle concentration are optionally used as a detergent forthe purposes of the present invention. Typically, the sample mixture isessentially cell-free. This method is not effective for detectingproteins that remain in cells or are associated with biologicalmembranes.

In another preferred embodiment the present dyes and staining solutionare employed to detect nucleic acids in a sample. Again, the stainingsolution is combined with the sample to form a dye-nucleic acid complexand then illuminated with an appropriate wavelength whereby the nucleicacid is detected.

Therefore, when used to stain nucleic acids or to form conjugates ofnucleic acids, the dye is combined with a sample that contains or isthought to contain a nucleic acid. The nucleic acid in the sample may beRNA or DNA, or a mixture or a hybrid thereof. Any nucleic acid isoptionally single- or multiple-stranded. The nucleic acid may be anatural polymer (biological in origin) or a synthetic polymer (modifiedor prepared artificially). The nucleic acid polymer (preferablycontaining at least 8 bases or base pairs) may be present as nucleicacid fragments, oligonucleotides, or larger nucleic acid polymers withsecondary or tertiary structure. The nucleic acid is optionally presentin a condensed phase, such as a chromosome, or in a protein complex,such as a ribosome or nucleosome. The nucleic acid polymer optionallycontains one or more modified bases or links or contains labels that arenon-covalently or covalently attached. For example, the modified base isoptionally a naturally occurring modified base, a known minor base, oris synthetically altered to contain an unusual linker such as morpholinederivatized phosphates, or peptide nucleic acids, or to contain a simplereactive functional group (<10 carbons) that is an aliphatic amine,carboxylic acid, alcohol, thiol or hydrazine, or to contain afluorescent label or other hapten. The sensitivity of the dyes forpolymers containing primarily modified bases and links may be diminishedby interference with the binding mode.

The nucleic acid may be present in solution, immobilized on a solid orsemi-solid matrix or present in a biological structure. Where theanalyte is enclosed in a biological structure, as is typical for nucleicacids, the sample is typically incubated with the dye. While permeantdyes of this class have shown an ability to permeate biologicalstructures rapidly and completely upon addition of the dye solution, anyother technique that is suitable for transporting the dye into thebiological structure is also a valid method of combining the sample withthe subject dye. Some cells actively transport the dyes across cellmembranes (e.g. endocytosis or ingestion by an organism or other uptakemechanism) regardless of their cell membrane permeability. Suitableartificial means for transporting the dyes (or pre-formed dye-nucleicacid complexes) across cell membranes include, but are not limited to,action of chemical agents such as detergents, enzymes or adenosinetriphosphate; receptor- or transport protein-mediated uptake; liposomesor alginate hydrogels; phagocytosis; pore-forming proteins;microinjection; electroporation; hypo-osmotic shock; or minimal physicaldisruption such as scrape loading, patch clamp methods, or bombardmentwith solid particles coated with or in the presence of the dyes.Preferably, where intact structures are desired, the methods forstaining cause minimal disruption of the viability of the cell andintegrity of cell or intracellular membranes. Alternatively, the cellsare fixed and treated with routine histochemical or cytochemicalprocedures, particularly where pathogenic organisms are suspected to bepresent. The cells are typically fixed immediately after staining withan aldehyde fixative that keeps the dye in the cells. In some cases,live or dead cells may even be fixed prior to staining withoutsubstantially increasing cell membrane permeability of previously livecells so that only cells that were already dead prior to fixation stainwith the cell-impermeant dye.

Typically, the biological structure that encloses the nucleic acid is acell or tissue, for example where the nucleic acid is present in a cellor interstitial space as a prokaryote or eukaryote microorganism, or asa virus, viroid, chromosome or organelle. Alternatively, the biologicalstructure is not enclosed in a tissue or cell, and is present either asa virus or as a microorganism or other cell, or is present as a cellularcomponent removed from its parent cell (e.g. a plasmid or chromosome, ora mitochondrion or nucleus or other organelle).

Typically, the biological structure is an organelle, chromosome or cellthat is optionally inside a eukaryote cell. The cell that is presentinside a eukaryote cell is typically a parasite or other infective agentsuch as a bacterium, protozoa, mycoplasma or mycobacterium. Where thenucleic acid is contained in a biological structure that is a cell, thecells are viable or dead cells or a mixture thereof, i.e. the integrityof the cell membrane is optionally intact or disrupted by natural(autolytic), mechanical or chemical means or by environmental means suchas changes in temperature or pressure. Alternatively, the cells areblebbing or undergoing apoptosis or necrosis or are in a cycle of growthor cell division.

Cell types for which the dye is an effective nucleic acid stain includecells with or without nuclei, including but not limited to, eukaryotes,such as plant and animal cells (particularly vertebrate cells),including pollen and gamete cells; prokaryotes, particularly bacteria,including both Gram-negative and Gram-positive bacteria; as well asyeast and other fungi, and spores. In one embodiment, the samplecomprises reticulocytes. The dyes are not equally effective in stainingall cell types and certain dyes are generally more permeant than others.Live cells are less permeable to the dyes than dead cells, andprokaryotes are typically less permeable than eukaryotes.

The nucleic acids in the sample, both natural and synthetic, may beobtained from a wide variety of sources. The presence of the nucleicacid in the sample may be due to natural biological processes, or theresult of a successful or unsuccessful synthesis or experimentalmethodology, undesirable contamination, or a disease state. The nucleicacid may be endogenous to the natural source or introduced as foreignmaterial, such as by infection, transfection, or therapeutic treatment,Nucleic acids may be present in all, or only part, of a sample, and thepresence of nucleic acids may be used to distinguish between individualsamples, or to differentiate a portion or region within a single sample,or to identify the sample or characteristics of the sample.

Typically, the sample containing nucleic acids is a cell or is anaqueous or aqueous miscible solution that is obtained directly from aliquid source or as a wash from a solid material (organic or inorganic)or a growth medium in which cells have been introduced for culturing ora buffer solution in which nucleic acids or biological structures havebeen placed for evaluation. Where the nucleic acids are in cells, thecells are optionally single cells, including microorganisms, or multiplecells associated with other cells in two or three dimensional layers,including multicellular organisms, embryos, tissues, biopsies,filaments, biofilms, etc.

Alternatively, the sample is a solid, optionally a smear or scrape or aretentate removed from a liquid or vapor by filtration. In one aspect ofthe invention, the sample is obtained from a biological fluid, includingseparated or unfiltered biological fluids such as urine, cerebrospinalfluid, blood, lymph fluids, tissue homogenate, interstitial fluid, cellextracts, mucus, saliva, sputum, stool, physiological secretions orother similar fluids. Alternatively, the sample is obtained from anenvironmental source such as soil, water, or air; or from an industrialsource such as taken from a waste stream, a water source, a supply line,or a production lot. Industrial sources also include fermentation media,such as from a biological reactor or food fermentation process such asbrewing; or foodstuffs, such as meat, grain, produce, or dairy products.

In yet another embodiment, the sample is immobilized in or on a solid orsemi-solid matrix wherein the nucleic acids of interest are present onor in the matrix. In one aspect of the invention, the matrix is amembrane. In another aspect, the matrix is an electrophoretic gel, suchas is used for separation and characterization of nucleic acids. Inanother aspect, the matrix is a silicon chip or glass slide, and thenucleic acids of interest have been immobilized on the chip or slide inan array.

Where the nucleic acid is present in a solution, the sample solution canvary from one of purified or synthetic nucleic acids such asoligonucleotides to crude mixtures such as cell extracts or homogenatesor other biological fluids, or dilute solutions from biological,industrial, or environmental sources. In some cases it is desirable toseparate the nucleic acids from a mixture of biomolecules or fluids inthe solution prior to combination with the dye. Numerous techniquesexist for separation and purification of nucleic acids from generallycrude mixtures with other proteins or other biological molecules. Theseinclude such means as chromatographic and electrophoretic techniques,using a variety of supports or solutions or in a flowing stream.Alternatively, mixtures of nucleic acids may be treated with RNase orDNase so that the polymer that is not degraded in the presence of thenuclease can be discriminated from degradation products using thesubject dyes.

The fluorescent dye-analyte complex is useful in essentially anyapplication previously described for other fluorescent nucleic acid orprotein stains (for example as described in U.S. Pat. No. 5,436,134 toHaugland et al. (1995); U.S. Pat. No. 5,445,946 to Roth et al. (1995);U.S. Pat. No. 5,534,416 to Millard et al, (1996); U.S. Pat. No.5,616,502 to Haugland et al. (1997).

Once the dye-analyte complex is formed, its presence may be detected andused as an indicator of the presence, location, or type of analyte inthe sample, or as a basis for sorting cells, or as a key tocharacterizing the sample or cells in the sample. Such characterizationmay be enhanced by the use of additional reagents, including fluorescentreagents. The amount of analyte in a sample can also be quantified bycomparison with known relationships between the fluorescence of thedye-analyte complex and concentration of analyte in the sample.

In one aspect of the invention, the fluorescence response of thedye-analyte complex is used as a means for detecting the presence orlocation of the analyte in the sample. The fluorescent signal isdetected by eye or by the instrumentation described above.

In addition, the staining profile that results from formation of thedye-analyte complex may be indicative of one or more characteristics ofthe sample. By staining profile is meant the shape, location,distribution, and spectral properties of the profile of fluorescentsignals resulting from excitation of the fluorescent dye-analytecomplexes. The sample can be characterized simply by staining the sampleand detecting the staining profile that is indicative of acharacteristic of the sample. More effective characterization isachieved by utilizing a dye that is selective for a certaincharacteristic of the sample being evaluated or by utilizing anadditional reagent (as described below), where the additional reagent isselective for the same characteristic to a greater or lesser extent orwhere the additional reagent is selective for a different characteristicof the same sample.

In one embodiment of the invention, where the dye is selected to bemembrane permeable or relatively impermeant to cell membranes, thestaining profile that results from the formation of the dye-analytecomplex is indicative of the integrity of the cell membrane, which inturn is indicative of cell viability. Alternatively, the more permeantdyes of the invention are used to stain both cells with intact membranesand cells with disrupted membranes, which when used in conjunction witha counterstain that gives a detectably different signal in cells withdisrupted membranes, allows the differentiation of viable cells fromdead cells. The counterstain that gives a detectably different signal incells with disrupted membranes is optionally an impermeant dye of theinvention or another reagent that indicates loss of integrity of thecell membrane or lack of metabolic activity of the dead cells.

In a further embodiment of the invention, the shape and distribution ofthe staining profile of dye-analyte complexes is indicative of the typeof cell or biological structure that contains the stained nucleic acids.Cells may be discriminated by eye based on the visual fluorescent signalor be discriminated by instrumentation as described above, based on thespectral properties of the fluorescent signal. Typically the stainingprofile used to characterize the sample is indicative of the presence,shape, or location of organelles or of cells, where the cells arelocated in a biological fluid, in a tissue, or in other cells.

In another aspect of the invention, the dye-analyte complex is used as afluorescent tracer or as a probe for the presence of a second analyte.In one aspect of the invention, the dye-analyte complex is used as asize or mobility standard, such as in electrophoresis or flow cytometry.Alternatively, the fluorescent signal that results from the interactionof the dye with the first analyte can be used to detect or quantitatethe activity or presence of other molecules that interact with the firstanalyte.

Applications of Chemically Reactive Cyanine Dyes

The reactive dyes of the invention are particularly useful forimmobilizing the dye on a surface or substrate, such as a polymericmicroparticle, polymeric membrane, fiber, or silicon chip. In thisembodiment, the dye is useful as a capture reagent for purification ordetection of an analyte, particularly nucleic acids. Alternatively, theimmobilized dyes are useful for moving associated analytes from oneenvironment to another via mechanical means. Where the subject dyes areattached to a surface, the surface can also function as a quantitativeor qualitative indicator of an analyte in solution, such as a test stripor dipstick.

The reactive dyes of the invention are also useful as detection reagentsduring the separation and purification of nucleic acids. For example thedyes of the invention can be conjugated to agar or agarose and used tocast plates or gels that are then useful for spot assays to quantitatenucleic acids. The ability of the reactive dyes of the invention to formstable covalent bonds with a variety of substrates allows the attachmentof nucleic acid stains to electrophoretic gel matrices (producing verystable and uniformly stained pre-prepared gels).

Alternatively, where nucleic acids are analyzed using capillaryelectrophoresis, the dyes of the invention may be conjugated in a small“band” or region in the capillary, resulting in detection of nucleicacids with high sensitivity, as the detecting reagent is concentratedinto a small volume, allowing efficient illumination using a laser orother excitation source. Where a conjugated dye is used in this fashion,the dye is optionally utilized only to capture the nucleic acids, withdetection accomplished by another detection reagent, or the fluorescentenhancement upon binding nucleic acids is utilized to detect the captureevent itself.

Where the dye is covalently bound to a substrate, such as a polymericmicroparticle or membrane, the nucleic acids that are associated withthe bound dye can be used as templates for translation and replication.Where the dye of the invention possesses a particularly high affinityfor nucleic acids, passing a solution containing nucleic acids over asuitable labeled polymer matrix results in depletion of nucleic acidsfrom the solution. The complexed nucleic acids can then be utilized inplace or extracted from the matrix for further analysis or utilization.

Alternatively, a reactive dye of the invention can be associated withpurified nucleic acids and covalently linked to the nucleic acids, whichare then transferred to a membrane by blotting, and covalently bound tothe polymeric membrane, resulting in a permanently labeled blot.

The dyes are optionally covalently bound to a molecule, such as adextran, which is normally excluded from live cells. When dead orpermeabilized cells then take up these molecules, the bound dyeinteracts with intracellular nucleic acids, giving rise to fluorescence.Similarly, the dye-conjugates of the invention possess utility forretaining conjugated substances within cells. This is particularlyuseful for conjugated substances that are typically excreted from livingcells relatively quickly, such as small organic molecules or by-productsof enzyme activity.

The dye is optionally conjugated to a substance selected such that theresulting conjugate has substantially different physical properties thanthose of the unbound dye, for example, conjugation of a dye of theinvention to a polysaccharide or a poly(ethylene glycol) polymer toinfluence solubility, cellular retention, or ability to bind analytes.

Applications of Covalently Labeled Nucleic Acids

The dyes of the invention are useful for labeling unmodified nucleicacids covalently (typically using a photoreactive dye) so that thelabeled nucleic acid retains its label through capillary transfer orelectrophoretic transfer to filter membranes and subsequenthybridization (for example as in Southern blotting). The dyes are alsouseful in Southwestern analysis, wherein the dye is first coupledcovalently to a nucleic acid, and the nucleic acid is then used to probea Western blot containing putative nucleic acid-binding proteins.Fluorescence enhancement indicates locations on the blot membrane wherethe nucleic acid has been captured by the immobilized protein.Conjugates of proteins that do not interact specifically with nucleicacids do not exhibit similar fluorescence enhancement when used atsimilar concentrations, and in the absence of detergent. Similarinteractions are useful for detecting immobilized DNA. Alternatively,where the protein is labeled with a second reagent, colocalization ofthat reagent and the dye of the invention indicates an interactionbetween the dye and the protein. This type of assay is also useful foranalyzing nucleic acid-binding drugs.

Where the dyes of the invention are used to covalently label nucleicacids, the labeled nucleic acid is useful as a probe to detectinteractions between nucleic acid-binding drugs or proteins, where thedrug or protein has been labeled with a second fluorophore or afluorescence quenching agent. Binding of the nucleic acid with the drugor protein results in loss of fluorescence. In this way, the nucleicacid-binding drug or protein can be quantitated, and the presence orabsence of either inhibitors or enhancers of nucleic acid binding couldbe identified. This application is also useful for in vivo or in vitrostudies, for example in the study of interactions between SSB (singlestrand binding protein) and single stranded DNA, histones and doublestranded DNA, sequence specific binding factors and their cognatesequences, or mismatch repair enzymes and mismatch regions of DNAhybrids.

Dye-labeled nucleic acids are useful for monitoring transfection intocells, either by the labeled nucleic acid itself, or where anothermolecule is cotransfected and the labeled nucleic acid is simply atracer. The amount of labeled nucleic acid in a cell could be used tostandardize the copy number of transiently transfected plasmids inreporter gene assays, such as chloramphenicol acetyltransferase assays(CAT assays).

Dye-conjugates of nucleic acids possess utility for detectinghybridization, as the dye-conjugate typically exhibits a change influorescence enhancement upon binding to a complementary strand ofnucleic acid. Dye-labeled nucleic acids are also useful for followingtriplex formation, or strand invasion, during DNA recombination. If twodyes capable of energy transfer are used, then real-time measurements ofhybridization or strand invasion/displacement may be made. In particularboth the interactions of an antisense oligonucleotides with nucleicacids and RNA splicing could be followed using this methodology.

Where the dye is used to covalently label a nucleic acid, the nucleicacid could then be ligated to another unlabeled nucleic acid, so thateven if the labeled nucleic acid fragment is unable to hybridize or bindproteins efficiently, the unlabeled portion retains full biologicalactivity. However, the nucleic acid is readily detectable due to thepresence of the attached dye.

The dyes of the invention also serve as haptens for secondary detection.The use of labeled antibodies directed against the dyes of the inventionallows for signal amplification in the detection of either a conjugatedsubstance, or a labeled nucleic acid. Alternatively, where theconjugated substance is a specific binding pair member, the specificbinding pair member may be used to amplify the detectable signal of thecyanine dye, typically by immunological methods. In this embodiment, theconjugated substance is typically a hapten, biotin or digoxigenin or anon-nucleic acid-binding dye. The dye-conjugate forms a nucleic acid-dyecomplex, producing enhancement of its fluorescence. The complex is thenlabeled with the complement of the specific binding pair member, whichis typically labeled with a fluorophore, producing an additionalfluorescent enhancement.

Additional Detection Reagents

The dyes of the invention can be used in conjunction with one or moreadditional reagents that are separately detectable. The additionalreagents may be separately detectable if they are used separately, e.g.used to stain different aliquots of the same sample, or if they staindifferent parts or components of a sample, regardless of whether thesignal of the additional reagents is detectably different from thefluorescent signal of the dye-analyte. Alternatively, the dye of theinvention is selected to give a detectable response that is differentfrom that of other reagents desired to be used in combination with thesubject dyes. Preferably the additional reagent or reagents arefluorescent and have different spectral properties from those of thedye-analyte complex. For example, dyes that form complexes that permitexcitation beyond 600 nm can be used in combination with commonly usedfluorescent antibodies such as those labeled with fluoresceinisothiocyanate or phycoerythrin. Any fluorescence detection system(including visual inspection) can be used to detect differences inspectral properties between dyes, with differing levels of sensitivity.Such differences include, but are not limited to, a difference inexcitation maxima, a difference in emission maxima, a difference influorescence lifetimes, a difference in fluorescence emission intensityat the same excitation wavelength or at a different wavelength, adifference in absorptivity, a difference in fluorescence polarization, adifference in fluorescence enhancement in combination with targetmaterials, or combinations thereof.

The detectably different dye is optionally one of the dyes of theinvention having different spectral properties and differentselectivity. In one aspect of the invention, the dye-analyte complex andthe additional detection reagents have the same or overlappingexcitation spectra, but possess visibly different emission spectra,generally having emission maxima separated by >10 nm, preferably >20 nm,more preferably >50 nm. Simultaneous excitation of all fluorescentreagents may require excitation of the sample at a wavelength that issuboptimal for each reagent individually, but optimal for thecombination of reagents. Alternatively, the additional reagent(s) can besimultaneously or sequentially excited at a wavelength that is differentfrom that used to excite the subject dye-analyte complex. In yet anotheralternative, one or more additional reagents are used to quench orpartially quench the fluorescence of the dye-analyte complex, such as byadding a second reagent to improve the selectivity for a particularnucleic acid or the AT/GC selectivity.

The additional dyes are optionally used to differentiate cells orcell-free samples containing the desired analyte according to size,shape, metabolic state, physiological condition, genotype, or otherbiological parameters or combinations thereof. The additional reagent isoptionally selective for a particular characteristic of the sample foruse in conjunction with a non-selective reagent for the samecharacteristic, or is selective for one characteristic of the sample foruse in conjunction with a reagent that is selective for anothercharacteristic of the sample. In one aspect of the invention, theadditional dye or dyes are metabolized intracellularly to give afluorescent product inside certain cells but not inside other cells, sothat the fluorescence response of the cyanine dye of the inventionpredominates only where such metabolic process is not taking place.Alternatively, the additional dye or dyes are specific for some externalcomponent of the cell such as cell surface proteins or receptors, e.g.fluorescent lectins or antibodies. In yet another aspect of theinvention, the additional dye or dyes actively or passively cross thecell membrane and are used to indicate the integrity or functioning ofthe cell membrane (e.g. calcein AM or BCECF AM). In another aspect, theadditional reagents bind selectively to AT-rich nucleic acids and areused to indicate chromosome banding, in another aspect of the invention,the additional reagent is an organelle stain, i.e. a stain that isselective for a particular organelle, for example the additionalreagent(s) may be selected for potential sensitive uptake into themitochondria (for example as described in U.S. Pat. No. 5,459,268 toHaugland et al. (1995)) or for uptake due to pH gradient in an organelleof a live cell (U.S. Pat. No. 5,869,689 to Zhang et al. (1999)).

The additional dyes are added to the sample being analyzed to be presentin an effective amount, with the optimal concentration of dye determinedby standard procedures generally known in the art. Each dye isoptionally prepared in a separate solution or combined in one solution,depending on the intended use. After illumination of the dyed cells at asuitable wavelength, as above, the cells are analyzed according to theirfluorescence response to the illumination. In addition, the differentialfluorescence response can be used as a basis for sorting the cells ornucleic acids for further analysis or experimentation. For example, allcells that “survive” a certain procedure are sorted, or all cells of acertain type in a sample are sorted. The cells can be sorted manually orusing an automated technique such as flow cytometry, according to theprocedures known in the art, such as in U.S. Pat. No. 4,665,024 toMansour, et al. (1987).

D. Kits of the Invention

In one aspect of the invention, the cyanine dyes of the invention areincorporated into a kit for commercial use. In one embodiment, the kitcomprises a selection of dyes of the invention, typically present as aconcentrated stock solution in a non-aqueous solvent, preferably DMSO.Where the kit is a “sampler” kit of cyanine dyes, the kit comprises 2-8distinct cyanine dyes, preferably 4-6 distinct cyanine dyes.

In another embodiment of the invention, the reagent kit comprises astock solution of the dye of the invention, typically present in a DMSOsolution; a buffer suitable for dilution of the stock solution, andoptionally further comprises a standard or an additional detectionreagent, or both. The standard is optionally one or more nucleic acids(for nucleic acid detection/quantification), or one or more proteins(for protein detection/quantification). The additional detection reagentis typically an organelle stain, a labeled immunoreagent, a drug, or anenzyme.

In yet another embodiment of the invention, the reagent kit comprises astock solution of the dye of the invention, typically present in a DMSOsolution, a buffer suitable for dilution of the stock solution, asilicon chip or glass slide, and nucleic acid standards.

A detailed description of the invention having been provided above, thefollowing examples are given for the purpose of illustrating theinvention and shall not be construed as being a limitation on the scopeof the invention or claims.

EXAMPLES Example 1 Preparation of aza-benzoxazole Precursors

The following 4-methyl-2-methylthiooxazolopyridinium tosylates areprepared by heating the corresponding 2-methylthiooxazolopyridines (M.Y. Chu-Moyer and R. Berger, J. Org. Chem. 60, 5721-5725 (1995)) with oneequivalent of methyl tosylate at 100-110° C. for one hour:

-   (4-methyl-2-methylthiooxazolo[4,5-b]pyridinium tosylate)

-   (6-methyl-2-methylthiooxazolo[5,4-c]pyridinium tosylate):

-   (1-methyl-2-methylthiooxazolo[5,4-b]pyridinium tosylate):

-   (4-methyl-2-methylthiooxazolo[4,5-b]quinolinium tosylate):

Example 2 Preparation of 5-methyl-2-methylthiothiazolo[4,5-b]pyridiniumtosylate (Compound 5)

To 0.626 g of 2-methylthiothiazolo[4,5-b]pyridine (Smith, K., Lindsay,C., Morris, I. K., Matthews, I. and Pritchard, G. J., Sulfur Letters 17,197-216 (1994)), is added 0.71 g methyl tosylate. The mixture is heatedat 120° C. for one hour. After cooling, the resulting oily mixture iswashed with 10 mL of ethyl acetate. After the ethyl acetate is decanted,Compound 5 is isolated as an oil.

Example 3 Preparation of 5-methyl-2-methylthiothiazolo[5,4-c]pyridiniumtosylate (Compound 6)

A mixture of 0.127 g of 2-methylthiothiazolo[5,4-c]pyridine (frommethylation of thiazolo[5,4-c]pyridine-2(1H)-thione with potassiumcarbonate and methyl iodide) and 0.143 g methyl tosylate is heated at120° C. for one hour. Ethyl acetate (8 mL) is added, the mixture isstirred for 30 minutes, and Compound 6 is isolated by suctionfiltration.

Example 4 Preparation of 3-methyl-2-methylthiothiazolo[5,4-b]pyridiniumtosylate (Compound 7)

A mixture of 1.285 g of 3-amino-2-chloropyridine, 0.48 g NaH (from ahexane-washed 60% dispersion of NaH in mineral oil), 0.6 mL CS₂ and 10mL DMF is heated at 110-120° C. for one hour to yieldthiazolo[5,4-b]pyridine-2(1H)-thione. The thione is methylated withpotassium carbonate and methyl iodide in DMF at room temperature to givethe methylthio derivative, 2-methylthiothiazolo[5,4-b]pyridine, which issubsequently heated with one equivalent of methyl tosylate at 100° C.for one hour to yield Compound 7.

Similarly, 6-chloro-4-methyl-2-methylthiothiazolo[4,5-b]pyridiniumtosylate is prepared from 6-chlorothiazolo[4,5-b]pyridine-2(1H)-thione:

-   6-bromo-4-methyl-2-methylthiothiazolo[4,5-b]pyridinium tosylate is    prepared from 2-amino-3,5-dibromopyridine:

-   4-methyl-2-methylthio-6-trifluoromethylthiazolo[4,5-b]pyridinium    tosylate is prepared from    2-amino-3-chloro-5-trifluoromethylpyridine:

-   and 1-methyl-2,7-bis(methylthio)thiazolo[5,4-d]pyridinium tosylate    is prepared from 5-amino-4,6-dichloropyrimidine:

Example 5 Preparation of3-acetoxy-1-methyl-2-acetylimino-1,2-dihydropyridine, p-toluenesulfonicacid salt (Compound 12)

2-Amino-3-hydroxypyridine (14.48 g) is triacetylated by heating with 3equivalents of acetic anhydride at 120-130° C. for 4 hours to yield,after silica gel column purification, 10.3 g of3-acetoxy-2-acetimidopyridine. This intermediate compound is heated for2 days at 65° C. with 3 equivalents of methyl tosylate to yield 7 g ofCompound 12.

Example 6 Preparation of 5-bromo-3-mercapto-2-methylaminopyridine(Compound 13)

Compound 13 is prepared by heating Compound 9 (1.99 g) with 30 mLmethanol and 15 mL of 10% NaOH at 80° C. for 8 hours. The pH is adjustedto 6-7 with acetic acid and the volume is reduced to ˜10 mL. The residueis filtered to yield 0.76 g of Compound 13.

Example 7 Preparation of Compound 14

To 0.55 g of Compound 1 and 0.49 g of 1,4-dimethylquinolinium tosylatein 10 mL of CH₂Cl₂ at room temperature is added 0.24 mL oftriethylamine. After 6 hours, the product is suction filtered to yield0.42 g of Compound 14.

Example 8 Preparation of Compounds 15, 16, 17, 18, 19, 20, 21, and 44

To a solution of 0.235 g of 4-methyl-1-phenyl-2-quinolone in 15 mL THFat −78° C., is added 0.6 mL of a 2.5 M solution of 1-butyl lithium inTHF. After 1 hr at −78° C., 1 mL of acetic acid is added. The reactionmixture is stirred for 1 hr at room temperature. The volatiles areevaporated and the residue is dissolved in 10 mL CH₂Cl₂. Compound 1 (0.7g) and 1 mL triethylamine are added. After 1 hr at room temperature thesolvent is evaporated, the crude product is dissolved in 10 mL ofmethanol and the solution is then added to 2.5 g NaI in 60 mL water. Theproduct, Compound 15, is filtered and further purified by columnchromatography on silica gel.

Similarly prepared except using phenyl lithium instead of butyl lithiumis Compound 16, which can be recrystallized from a mixture of DMF andethyl acetate.

Compound 17 is prepared similarly from 4-methyl-1-phenyl-2-quinolone

Compound 18 is prepared similarly from1-ethyl-6,7-methylenedioxy-4-methyl-2-quinolone

Compound 19 is prepared similarly from6-methoxy-1,4-dimethyl-2-quinolone

Compound 20 is prepared similarly from6,7-dimethoxy-1,4-dimethyl-2-quinolone

Compound 21 is prepared similarly from7-methoxy-1,4-dimethyl-2-quinolone.

Compound 44 is prepared similarly, except that Compound 9 is usedinstead of Compound 1:

Example 9 Preparation of Compound 22

To 0.605 g of 4′-bromobenzyldiethylamine in 10 mL of THF at −78° C.under nitrogen is added 0.8 mL of a 2.5 M butyl lithium in THF, followedby a solution of 0.173 g of 1,4-dimethyl-2-quinolone in 10 mL of THF.After one hour at −78° C., 1 mL of acetic acid is added. After warmingto room temperature for another hour the volatiles are evaporated andthe residue is dissolved in 10 mL CH₂Cl₂. To this solution is added 0.67g of Compound 2, followed by 1 mL triethylamine. After one hour at roomtemperature the volatiles are evaporated and the residue is dissolved in7 mL of methanol. This solution is added to a solution of 2.25 g NaI and0.8 g NaOH in 50 mL water. Compound 22 is filtered, dried andrecrystallized from a mixture of DMF and ethyl acetate.

Compound 23 is prepared similarly, using6-methoxy-1,4-dimethyl-2-quinolone.

Example 10 Preparation of Compound 24

The compound is prepared by heating 0.1 g of Compound 22 in 5 mL DMFwith 0.5 mL methyl iodide at 60° C. in a sealed tube for 2 hours. Afterthe reaction mixture is cooled to room temperature, 5 mL ethyl acetateis added and Compound 24 is collected by suction filtration.

Compound 25 is similarly obtained from Compound 23.

Example 11 Preparation of Compounds 26 and 27

A mixture of 1.06 g of Compound 1, 0.88 g of1-(3′-iodopropyl)-4-methylquinolinium iodide (U.S. Pat. No. 5,321,130 toYue et al. (1994)), 15 mL CH₂Cl₂ and 0.28 mL triethylamine is stirred atroom temperature for 30 minutes. The volatiles are evaporated and theresidue is dissolved in 10 mL methanol. The resulting solution is addedto 4.5 g NaI in 60 mL water. Compound 26 is obtained by suctionfiltration. A solution of Compound 26 (0.15 g) and 1 mL of a 25%trimethylamine in methanol solution are heated in a sealed tube at75-80° C. for 7 hours. Following cooling and filtration, Compound 27 isrecrystallized from a mixture of DMF and CH₂Cl₂.

Compound 47 is similarly prepared from Compound 9

Example 12 Preparation of Compound 28

A mixture of 0.2 g of Compound 26, 15 mL DMF and 0.029 mLN,N,N′,N′-tetramethylpropanediamine is heated at 80° C. for 4 days. Tothe solution is added 6 mL of chloroform and Compound 28 is isolated bysuction filtration.

Example 13 Preparation of Compounds 45 and 46

A mixture of 2.1 g of Compound 9, 1.5 g of1-(3′-iodopropyl)-4-methylquinolinium iodide, 0.7 mL triethylamine and30 mL CH₂Cl₂ is stirred at room temperature for 2 hours. Followingevaporation, the residue is dissolved in 30 mL DMF and added to 8 g NaIin 200 mL water. Compound 45 is obtained by suction filtration. Compound45 (1 g), 0.125 mL of N,N,N′,N′-tetramethylpropanediamine and 8 mL DMFare heated at 80° C. for 4 days. Following cooling, 24 mL of chloroformis added and Compound 46 is purified by recrystallization for a mixtureof DMF and methanol.

Example 14 Preparation of Compounds 29, 30, and 41

A mixture of 6 g of Compound 1, 3 g of 4-methyl-1-phenyl-2-quinolone,8.9 mL diisopropylethylamine, and 1.2 mL of trimethylsilyltrifluoromethanesulfonate is heated at reflux in 100 mL of CH₂Cl₂ for 1hour. The reaction mixture is cooled on ice and 100 mL of water is addedslowly with stirring. The layers are separated and the aqueous layer isextracted with chloroform. The combined organic layers are washed withbrine and dried over Na₂SO₄. The crude4-oxazolopyridinylmethylidenequinolone thus obtained is heated at refluxfor 3 hours with 5.8 mL of phosphorus oxychloride and 100 mL ofdichloroethane. The reaction mixture is cooled to room temperature, 100mL of water is added and after 30 min, the crude product is recovered byfiltration. The crude product is purified by chromatography on a silicagel column to yield Compound 29. A mixture of 0.1 g of Compound 29, 10mL of dichloroethane and 0.27 mL of diethylamine is heated at 60° C. ina sealed tube for 2.5 hours. The volatile components are evaporated andthe crude residue is purified by chromatography on a silica gel columnto yield Compound 30.

Preparation of Compound 41 is similar to that of Compound 29, exceptthat Compound 9 is used instead of Compound 1.

Example 15 Preparation of Compound 31

Triethylamine (0.1 mL) is added to 0.176 g of Compound 11 and 0.165 g of1,4-dimethylquinolinium tosylate in 10 mL of CH₂Cl₂ at room temperature.After stirring for one hour, the organic layer is evaporated and theresidue is dissolved in 2 mL of methanol and added dropwise to asolution of 1.5 g NaI in 25 mL of water. Compound 31 is recovered bysuction filtration.

Example 16 Preparation of Compound 32

A mixture of 0.35 g of 1,4-dimethyl-2-quinolone, 0.56 mL phosphorusoxychloride, and 0.05 mL DMF is heated in 10 mL toluene at 70° C. for 2hours. After cooling, 20 mL CH₂Cl₂ and 1.11 g 2-mercaptopyridine areadded and the reaction mixture is heated overnight at 40° C. Thevolatiles are evaporated and to the residue is added 0.84 g of Compound1, 1.4 mL of triethylamine and 20 mL CH₂Cl₂. After 2 hours, thevolatiles are evaporated, the residue is dissolved in 3 mL methanol andthe solution is added to 4.5 g NaI in 20 mL water. Compound 32 ispurified by recrystallization from a mixture of DMF and ethyl acetate.

Example 17 Preparation of Compound 33

A mixture of 0.244 g of Compound 7, 0.225 g of 1,4-dimethylquinoliniumtosylate, 0.14 mL triethylamine and 10 mL CH₂Cl₂ is stirred at roomtemperature overnight. The volatiles are evaporated, residue isdissolved in 4 mL of methanol, and the resulting solution is added to1.5 g NaI in 25 mL water. Compound 33 is obtained by suction filtration.

Compounds 34, 35, 36, 37, 38, 39, and 40 are similarly prepared from1,4-dimethylquinolinium tosylate and Compounds 6, 11, 4, 10, 5, 9, and 8respectively.

Example 18 Preparation of Compound 42

A mixture of 0.2 g of Compound 8, 0.065 mL of lepidine, 0.28 mL aceticanhydride and 5 mL DMF is heated at 110-120° C. for one hour. Thereaction mixture is carefully diluted with a mixture of sodiumbicarbonate and water and then extracted with chloroform. The chloroformis evaporated, and the resulting residue is purified by silica gelchromatography to yield Compound 42.

Compound 43 is similarly prepared from Compound 1.

Example 19 Preparation of Compound 48

Compound 1 (0.36 g), 0.28 g of 1,4-dimethylpridinium tosylate, 0.17 mLof triethylamine and 20 mL of CH₂Cl₂ are stirred at room temperature forone hour, then stirred for an additional 3 days at −20° C. Compound 48is isolated by suction filtration.

Example 20 Preparation of Compound 49

A solution of 4.2 mg of3-methyl-2-methylhio-4,5,7-trifluorothiazolo[5,4-c]pyridinium tosylate(from 4-amino-2,3,5,6-tetrafluoropyridine by a procedure similar to thatused for preparation of Compound 8), 3.3 g of 1,4-dimethylquinoliniumtosylate and 1.7 mL of triethylamine is stirred in 20 mL CH₂Cl₂ at roomtemperature for 1 hour. The volatiles are evaporated and the residue ispurified by chromatography on a silica gel column. The product isconverted to Compound 49 with NaI in water.

Example 21 Preparation of Compound 50

A mixture of 0.33 g of 1,4-dimethylquinolinium tosylate, 0.24 g ofN,N′-diphenylformamidine, and 0.3 mL acetic anhydride is heated at 130°C. for 2 hours. Ethyl acetate (20 mL) is added and the solution isheated at reflux for 20 minutes. The mixture is cooled, and theresulting solid is dissolved in 5 mL dichloroethane. Compound 12 (0.34g), 0.35 mL of diisopropylethylamine and 0.2 mL acetic anhydride areadded. After stirring for 3 hours at room temperature, the volatiles areevaporated, the residue is dissolved in 3 mL of methanol and thesolution is added dropwise to 1.5 g NaI in 20 mL water. Compound 50 isrecovered by filtration and purified by chromatography on a silica gelcolumn.

Example 22 Preparation of Compound 51

A mixture of 1.9 g of Compound 12, 1.18 g of N,N′-diphenylformamidine,2.1 mL of triethylamine and 50 mL dichloroethane is heated at 60-65° C.for 2 hours. After cooling, one equivalent of1,4-dimethyl-2-phenylquinolinium salt (prepared in a manner similar tothat for Compound 16) in 10 mL of dichloroethane is added, followed by0.7 mL of additional triethylamine and 1.41 mL of acetic anhydride.After three hours, the volatiles are evaporated, the residue isdissolved in 10 mL DMF and the solution is added to 7.5 g of NaI in 120mL water. Compound 51 is recrystallized from a mixture of methanol andethyl acetate.

Example 23 Preparation of Compound 52

A mixture of 0.18 g of Compound 2, 0.17 g 1,4-dimethylpyridiniumtosylate, 0.09 mL triethylamine and 20 mL CH₂Cl₂ is stirred at roomtemperature overnight. The volatiles are evaporated, the residue isdissolved in 2 mL of MeOH and added to 1.5 g NaI in 12 mL water. Theaqueous layer is extracted with 1-butanol and Compound 52 is purified bychromatography on a silica gel column.

Example 24 Preparation of Compound 53

Compound 1 (0.35 g), 0.33 g of 1,2-dimethylquinolinium tosylate, 0.15 mLof triethylamine and 20 mL CH₂Cl₂ are stirred at room temperature forone hour. The reaction is suction filtered to yield Compound 53, whichis purified by chromatography on silica gel.

Example 25 Preparation of Compound 54

A mixture of 0.173 g of 1,4-dimethyl-2-quinolone, 0.28 mL phosphorusoxychloride, 0.025 mL DMF and 5 mL toluene is heated at 70° C. for 6hours. Following cooling, 10 mL of methanol is added and the solution isheated at 45° C. for two hours. The volatiles are evaporated, 0.352 g ofCompound 1, 0.7 mL of triethylamine and 20 mL of CH₂Cl₂ are added andthe mixture is stirred overnight at room temperature. The solvent isevaporated, the crude product is dissolved in 3 mL methanol and added to2.5 g of sodium perchlorate in 20 mL water. Compound 54 is collected byfiltration, dried, and recrystallized from a mixture of DMF and ethylacetate.

Example 26 Preparation of Compound 55

Compound 13 (55 mg) is stirred with 10 mg sodium borohydride for 1.5hours. Acetic acid (0.6 mL) is added to quench the reaction. Thevolatiles are evaporated. The crude product is stirred with 0.21 mLtriethylamine, 0.19 mL acetic anhydride and 10 mL CH₂Cl₂. After one hourat room temperature and 30 minutes at 50-60° C., the volatiles areevaporated. Ethyl acetate (10 mL) is added, followed by 5 mL of hexanes.The solid is filtered and the salt thus obtained is stirred overnight atroom temperature with a mixture of 65 mg N,N′-diphenylformamidine and0.1 mL triethylamine in 4 mL dichloroethane. To this is added 150 mg of1,4-dimethyl-2-phenylquinolinium derivative in 10 mL of acetonitrile,another 0.1 mL of triethylamine and 0.07 mL acetic anhydride. After 1.5hr at room temperature the volatiles are evaporated, the residue isdissolved in 3 mL methanol and the solution is added to 1 g NaI in 20 mLwater. Compound 55 is isolated by suction filtration followed bypurification on a silica gel column.

Example 27 Preparation of Compound 56

A solution of 1.14 g of Compound 12 in 30 mL of dichloroethane and 1.7mL triethylamine is stirred at room temperature for 30 minutes. To thisis added 0.7 g of N,N′-diphenylformamidine and the mixture is heated at65° C. for 4 hours. Following cooling, 0.84 g of1-(4′-sulfobutyl)-4-methylquinolinium inner salt is added, followed by0.6 mL of acetic anhydride. The mixture is stirred for 30 minutes andCompound 56 is collected by suction filtration.

Example 28 Preparation of Compound 57

To 4-ethyl-1-methyl-2-quinolone in THF at −78° C. is added 1.5equivalent of phenyllithium, and the resulting solution is kept cold andstirred for one hour. Four equivalents of acetic acid are introduced andthe reaction mixture is allowed to stir at room temperature for 3 hours.All the volatile components are then removed under reduced pressure. Theresulting residue is dissolved in methylene chloride and one equivalentof Compound 1 (4-methyl-2-methylhiooxazolo[4,5-b]pyridinium tosylate) isintroduced followed by 5 equivalents of triethylamine. The resultingproduct is purified by column chromatography on silica gel.

Example 29 Preparation of Compound 58

To 2,4-dimethyl-thiazolo-[4,5-b]pyridinium tosylate in pyridine at roomtemperature is added 1.2 equivalent of benzoyl chloride, and theresulting mixture is stirred at room temperature overnight. Theresulting intermediate is isolated by silica gel column chromatography.This intermediate is then heated at reflux in 3 equivalents ofphosphorous oxychloride for 3 hours. Excess phosphorous oxychloride isremoved under reduced pressure and the residue is stirred in methylenechloride with one equivalent of 1,4-dimethyl-quinolinium tosylate and 4equivalents of triethylamine to obtain the desired product.

Example 30 Preparation of Compound 59

To a mixture of one equivalent each of 1,4-dimethylquinolinium tosylateand malonaldehyde dianil hydrochloride in acetic acid, is added oneequivalent of acetic anhydride and the resulting reaction mixture isheated at 150° C. for 30 minutes. All volatile components are removedunder reduced pressure and methylene chloride is introduced followed bythe addition of one equivalent of2-ethyl-4-methyloxazolo[4,5-b]pyridinium tosylate, 1.5 equivalent ofacetic anhydride, and 3 equivalents of triethylamine to obtain thedesired product.

Example 31 Preparation of Compound 60

A mixture of 135 mg of2-benzoylmethylidene-3-methyl-2,3-dihydrobenzothiazole and 230 mg ofphosphorous oxychloride is heated at reflux in 5 mL of dichloroethanefor 2 hours. Solvent and excess reagents are evaporated under reducedpressure and 30 mL of ethyl acetate is added. After stirring for 30minutes at room temperature, the resulting solid is collected byfiltration and resuspended in 15 mL of methylene chloride. To themixture is added 165 mg of 1,4-dimethylquinolinium tosylate and 0.14 mLof triethylamine. After stirring at room temperature overnight, thedesired product is recovered by recrystallization. When associated withDNA, Compound 60 exhibits excitation and emission wavelengths of 642 nmand 659 nm, respectively.

Example 32 Detecting Double-Stranded DNA in Solution

Samples containing 0.1 ng/mL to 1 microgram/mL double-stranded DNA areprepared in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. The selected dye of theinvention is dissolved in DMSO at a concentration of 1-10 mM and thendiluted into the same buffer at a concentration sufficient to achievefinal optical densities of 0.1 and 0.01 at 500 nm, 530 nm, 540 nm or 560nm. Equal volumes (100 microliters each) of DNA sample solution and dyesolution at each concentration are combined and mixed gently at roomtemperature in a microplate well.

Samples are allowed to equilibrate for 5-60 minutes, protected fromlight, and the fluorescence of the sample is measured using afluorescence microplate reader. DNA concentration is determined bycomparison of the sample fluorescence with a standard curve obtainedwith that dye for each dye concentration. The sensitivity limit ofseveral dyes in this assay is shown (Table 5).

TABLE 5 Detecting double-stranded DNA in solution. Sensitivity Limit(ng/ml) Low dye concentration High dye concentration Dye (0.01 OD) (0.1OD) Excitation 500 nm/Emission 560 nm Compound 43 10 10 Compound 48 >1010 Compound 49 >10 20 Compound 53 >10 10 Compound 54 1.0 10 Excitation530 nm/Emission 560 nm Compound 14 1.0 <10 Compound 15 0.5 10 Compound17 2.0 <10 Compound 18 0.2-0.5 <10 Compound 20 0.2 10 Compound 21 0.2<10 Compound 27 0.5 20 Compound 28 0.2 <10 Compound 30 1.0 10 Compound31 2.0 50 Compound 32 1.0 10 Excitation 540 nm/Emission 570 nm Compound16 1.0 <10 Compound 19 0.5 <10 Compound 22 1.0 10 Compound 23 0.2 <10Compound 24 5.0 10 Compound 25 0.5 <10 Compound 29 2.0 100 Compound33 >10 100 Compound 34 0.2 <10 Compound 35 0.5 10 Excitation 560nm/Emission 585 nm Compound 37 2.0 10 Compound 38 5.0 200 Compound 395.0 10 Compound 40 1.0 10 Compound 41 >10 10 Compound 42 >10 10

Example 33 Detecting Oligonucleotides in Solution

Selected dyes of the invention are dissolved in DMSO at 1-10 micromolarconcentration, then diluted into 10 mM Tris-HCl, 1 mM EDTA, pH 7.5 to afinal optical density of 0.1 or 0.01 at 530 nm or 540 nm. The resultingdye solutions are combined with an equal volume of an 18-baseoligodeoxynucleotide sequencing primer solution at concentrations of 0.1ng/mL to 1 microgram/mL in the same buffer. The resulting mixture ismixed gently and incubated for 5-60 minutes at room temperature,protected from light, and the sample fluorescence is measured in afluorescence microplate reader. Typical sensitivity limits for some dyesare shown in Table 6.

TABLE 6 Detection of oligonucleotides in solution. Sensitivity Limit(ng/mL) Low dye concentration High dye concentration Dye (0.01 OD) (0.1OD) Excitation 530 nm/Emission 560 nm Compound 18 >10 <10 Compound 20 10<10 Compound 28 >10 <10 Excitation 540 nm/Emission 570 nm Compound 231.0 <10 Compound 25 >10 <10 Compound 19 >10 <10

Example 34 Detecting RNA in Solution

Selected dyes of the invention are dissolved in DMSO at 1-10 micromolarconcentration, then diluted into 10 mM Tris-HCl, 1 mM EDTA, pH 7.5 to afinal optical density of 0.1 or 0.01 at 530 nm or 540 nm. These dyesolutions are combined with an equal volume of Escherichia coliribosomal RNA at concentrations ranging from 0.25 ng/mL to 1microgram/mL in microplate wells. The resulting mixtures are mixedgently and incubated for 5-60 minutes at room temperature, protectedfrom light, and the sample fluorescence is measured. Typical sensitivitylimits for selected dyes are shown in Table 7.

TABLE 7 Detecting RNA in solution. Sensitivity Limit (ng/mL) Low dyeconcentration High dye concentration Dye (0.01 OD) (0.1 OD) Excitation530 nm/Emission 560 nm Compound 18 1.2 10 Compound 20 5.0 200 Compound28 >10 500 Excitation 540 nm/Emission 570 nm Compound 19 10 20 Compound23 2.5 200 Compound 25 1.2 200

Example 35 Detecting Double-Stranded DNA in the Presence ofSingle-Stranded DNA

Selected dyes of the invention exhibit high sensitivity for detectingdouble-stranded DNA, but exhibit little sensitivity for detectingsingle-stranded nucleic acids, making them particularly useful forquantitating double-stranded DNA in the presence of single-stranded DNA,RNA or oligonucleotide primers (such as might be present in a PCRreaction). Samples containing nucleic acids at a concentration of 2micrograms/mL are combined with Compound 16, Compound 22, or Compound 24at a final dye concentration of 1 micromolar in 10 mM Tris-HCl, 1 mMEDTA, pH 7.5, and mixed gently. The resulting mixture is allowed toincubate for 5-60 minutes at room temperature, protected from light, andsample fluorescence is measured using a fluorescence microplate reader,with excitation at 540 nm and emission at 570 nm. Double-stranded DNA isdetected preferentially (FIGS. 1 and 2). If, however, the dyeconcentration is reduced relative to the concentration ofsingle-stranded nucleic acids, the fluorescence upon binding thosenucleic acids increases relative to that of the double-stranded DNA-dyecomplex. Thus the assay is always performed in sufficient dye excess.

Example 36 Detecting Nucleic Acids in Gels

DNA samples are loaded onto agarose or polyacrylamide gels andelectrophoresed using standard conditions. Selected dyes of theinvention are dissolved in DMSO at a concentration of 1-10 micromolarand diluted into 0.5×TBE buffer to a final concentration of 1 micromolardye. Gels are incubated for 20-90 minutes in this staining solution,with gentle agitation, at room temperature, protected from light.Nucleic acids are visualized by transillumination at 300 nm, followed bydirect visual detection or photography using Polaroid black-and-whiteprint film. Nucleic acids are alternatively visualized by scanning usinga FMBIO II laser scanner with a 532 nm frequency-doubled YAG lasersource or a charge-coupled device camera system coupled with anultraviolet (300 nm) light source, or a visible light transilluminator.The sensitivity of detection obtained using these dyes and variousimaging instruments is shown in Table 8.

TABLE 8 Sensitivity for detecting double-stranded bacteriophage λ cl857DNA that is digested with HindIII restriction endonuclease, andseparated in agarose gels. Imaging system POLAROID 667 POLAROID 667BOEHRINGER MANNHEIM HITACHI FMBIO II B&W film B&W film LUMIIMAGER CCDlaser scanner Excitation FOTODYNE FOTODYNE DARK READER 300 nm trans. 300nm trans. 532 nm freq.-doubled source 300 nm trans. 254 nm epi. visiblelight box 520 nm em. 545 nm em YAG laser Cpd. 15 240 pg 60 pg 480 pg 210pg 100 pg 100 pg  Cpd. 16 120 pg 30 pg 240 pg 210 pg  50 pg 25 pg Cpd.18 120 pg 60 pg nd 210 pg 100 pg 50 pg Cpd. 20 480 pg 120 pg  nd 830 pg210 pg 50 pg Cpd. 21 120 pg 60 pg nd 420 pg  50 pg 50 pg Cpd. 22 120 pg30 pg  60 pg 830 pg  50 pg  7 pg Cpd. 23 120 pg 60 pg 240 pg 830 pg 100pg  5 pg Cpd. 24  60 pg 30 pg 120 pg 420 pg  50 pg  5 pg Cpd. 25 120 pg120 pg  240 pg  1.7 ng 100 pg 25 pg nd indicates that a value was notdetermined for this dye with this gel imager.

Example 37 Detecting Nucleic Acids Arrayed on Glass Slides

Nucleic acid arrays are prepared on glass slides, using an automatedarraying robot. The slide surface is optionally blocked with a solutionof 0.1% Ficoll (Type 400, Amersham Pharmacia) and 0.1%polyvinylpyrrolidone. Arrays are allowed to dry and then overlaid with asolution containing 1 micromolar dye in 10 mM Tris-HCl, 1 mM EDTA, pH7.5. Suitable dyes for this purpose include Compound 15, Compound 16,Compound 22, Compound 23, Compound 24, and Compound 25. Slides areincubated with the overlay solution for 5-60 minutes at roomtemperature, with light protection, and then are washed briefly in thesame buffer without the dye. The amount of nucleic acid in each spot onthe array can be measured by comparison of the fluorescence of anunknown sample with that of standards of known concentration, which areapplied to the same slide. Fluorescence measurement is performed using afluorescence microscope with a mercury or xenon lamp, equipped withappropriate optical filters, a charge-coupled device and image analysissoftware, or using a dedicated array reader with a 532 nm or 546 nmexcitation source, such as the GENEARRAY 2000 (General Scanning).

Example 38 Detecting Proteins in Solution

Compound 51 and Compound 44 are dissolved in DMSO at 1-10 mM and thendiluted into 10 mM Tris-HCl, 1 mM EDTA, pH 7.5, containing 0.05% sodiumdodecyl sulfate, to a final concentration of 2 micromolar. This dyesolution is combined with samples containing bovine serum albumin at 100ng/mL to 10 micrograms/mL. Samples are optionally incubated for 10minutes at 90° C. and then cooled to room temperature, or are incubatedfor 5-60 minutes at room temperature. Fluorescence is measured using afluorescence microplate reader with excitation at 540 nm and emission at590 nm for Compound 44 and with excitation at 600 nm and emission at 670nm for Compound 51. The fluorescence signal obtained from Compound 44 or51 in buffer alone (control) is subtracted from the fluorescencerecorded for the protein-containing samples in order to determine theintensity of the signal that is due to the presence of protein. Proteinconcentrations are then determined by comparison of the signal intensitythus obtained with the signals obtained using a dilution series of knownconcentration prepared using either the same protein or a proteinstandard, such as bovine serum albumin. The sensitivity limit for eitherdye under either condition is about 100 ng/mL bovine serum albumin(FIGS. 3 and 4).

Example 39 Detection of Proteins in Sodium Dodecyl Sulfate(SDS)-Polyacrylamide Gels

The pure protein or mixture of proteins of interest is prepared inLoading Buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 0.015%bromophenol blue). Dithiothreitol is added to each sample, to a finalconcentration of 0.1 M. The samples are heated for 4-5 minutes at 90-95□C and loaded onto a 15% Tris-glycine polyacrylamide mini- or full-sizedgel containing 0.05%-0.1% SDS, with a 4% stacking gel. The gel iselectrophoresed under standard conditions, in a standard Tris-glycinebuffer containing 0.05%-0.1% SDS. The resulting gel is transferred to asmall staining dish containing a 1-3 μM solution of Compound 25 in 7.5%acetic acid (in water). The staining solution is then covered with foilto protect it from room light and gently agitated for 45 minutes-1 hour.After 10 minutes, the protein bands are readily apparent, butsensitivity improves over about 30-40 minutes. The gel is then removedfrom the staining dish, rinsed briefly in 7.5% acetic acid andtransferred directly to a UV-transilluminator. The gel, with the stainedprotein bands is photographed using 300 nm transillumination and blackand white POLAROID 667 print film with a Wratten 9 gelatin filter. Thestained bands appear visually as brightly fluorescent orange bands.Proteins appear as white bands on a grey to black background in thePOLAROID photograph.

Although Compound 25 is used in this procedure, a variety of other dyesof the invention are useful as electrophoretic gel stains, yieldingstained gels that possess bands having fluorescence emission from yellowto red.

Example 40 Detection of Apoptosis

A 1 mM solution of Compound 27 in DMSO is prepared. Jurkat human T-cellleukemia cells are treated with 10 μM camptothecin to induce apoptosis.A sample of the cells (1×10⁸ cells/mL) is incubated with enough of theCompound 27 stock solution to give a final dye concentration of 0.1 μM,and sufficient propidium iodide to give a final concentration of 1.5 μM.After incubating for 30 minutes on ice, the cell samples are analyzed ona FacsVantage flow cytometer using 488-argon laser excitation, withemission at 530/30 and 675/20. Apoptotic cells are compared to controlcells that were incubated with the same final concentration of DMSO usedin the staining solution. A plot of FL1 versus FL3 shows that Compound27 is capable of differentiating between apoptotic and normal cells.Late stage apoptotic and necrotic cells are stained with propidiumiodide.

It is to be understood that, while the foregoing invention has beendescribed in detail by way of illustration and example, numerousmodifications, substitutions, and alterations are possible withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A method for detecting double-stranded DNA in thepresence of single-stranded nucleic acid in a sample, wherein the methodcomprises the steps: a) combining the sample with a staining solution,wherein the staining solution comprises one or more dye compounds havinga structure according to

wherein X is O, S, Se, NR¹⁵, or CR¹⁶R¹⁷, where R¹⁵ is H or an alkylgroup having 1-6 carbons; and R¹⁶ and R¹⁷, which may be the same ordifferent, are independently C₁-C₆ alkyl groups, or R¹⁶ and R¹⁷ taken incombination complete a 5- or 6-membered saturated ring; R² is selectedfrom the group consisting of -L-Rx, -L-Sc, TAIL, BRIDGE and a C₁-C₆alkyl group that is optionally substituted by sulfo, carboxy, amino,substituted amino or substituted ammonium, wherein α is 0 or 1 and β is0 or 1 provided that α+β=1; and TAIL is a heteroatom-containing moiety;wherein Rx is a reactive group; Sc is a conjugated substance; and L andBRIDGE are independently a single covalent bond, or a covalent linkage;Y is —CR³═CR⁴—wherein p and m=0 or 1, such that p+m=1; R³, R⁴, R⁶, andR⁷ are independently selected from the group consisting of hydrogen, aC₁-C₆ alkyl, a halogen, a CYCLIC SUBSTITUENT, —OR⁸, —SR⁸, —(NR⁸R⁹),TAIL; BRIDGE, -L-Rx and -L-Sc; where R⁸ and R⁹ are independently a C₁-C₆alkyl group, 1-2 alicyclic or aromatic rings; or R⁸ and R⁹ taken incombination are —(CH₂)₂—V—(CH₂)₂—where V is a single bond, —O—, —CH₂—,or —NR¹⁰—, where R¹⁰is H or a C₁-C₆ alkyl; wherein CYCLIC SUBSTITUENT isa substituted or unsubstituted aryl, heteroaryl or C₃-C₁₀ cycloalkyl; orR⁶ and R⁷ form a fused aromatic ring —R¹¹═R¹²—R¹³═R¹⁴—wherein R¹¹, R¹²,R¹³, and R¹⁴ are independently selected from the group consisting ofhydrogen, C₁-C₆ alkyl group, —OR⁸, —SR⁸, —(NR⁸R⁹), a CYCLIC SUBSTITUENT,TAIL, BRIDGE, -L-Rx and -L-Sc; R⁵ is independently selected from thegroup consisting of a C₁-C₆ alkyl group, a CYCLIC SUBSTITUENT, TAIL,BRIDGE, -L-Rx and -L-Sc; or R⁵ is absent; R³⁰, R³¹, and R³² areindependently selected from the group consisting of hydrogen, C₁- C₆alkyl, C₃-C₁₀ cycloalkyl, aryl, and heteroaryl, wherein n=0, 1 or 2;wherein BRIDGE, when present, is bound to the dye compound or anotherunsymmetrical cyanine dye; b) incubating the sample and the stainingsolution for a sufficient amount of time to form a dye-nucleic acidcomplex; c) illuminating the complex with an appropriate wavelengthwhereby the double-stranded DNA is detected in the presence ofsingle-stranded nucleic acid.
 2. The method according to claim 1,wherein X is O, n is 0, R² is methyl, and R⁶ and R⁷ taken together forma fused aromatic ring -—R¹¹═R¹²—R¹³═R¹⁴—wherein R¹¹, R¹², R¹³, and R¹⁴are independently selected from the group consisting of hydrogen, C₁-C₆alkyl, and —OR⁸.
 3. The method according to claim 1, wherein R⁵ is aCYCLIC SUBSTITUENT that is an aryl group and R⁴ is a C₁-C₆ alkyl group.4. The method according to claim 1, wherein R⁵ is a C₁-C₆ alkyl groupand R⁴ is a CYCLIC SUBSTITUENT that is an aryl group.
 5. The methodaccording to claim 1, wherein the dye is substituted by at least oneTAIL substituent.
 6. The method according to claim 5, wherein the TAILis according to formula LINK-SPACER-CAP; wherein LINK is a singlecovalent bond, an ether linkage (—O—), a thioether linkage (—S—), or anamine linkage (—NR²⁰—); where R²⁰ is hydrogen, a C₁-C₈ alkyl group orSPACER-CAP; SPACER is a covalent linkage; and CAP is —OR²¹, —SR²¹,—NR²¹R²², or —NR²¹R²²R²³; where R²¹, R²², and R²³ are independentlyselected from the group consisting of hydrogen, a C₁-C₈ alkyl, and aC₁-C₈ cycloalkyl wherein the alkyl or cycloalkyl are optionallysubstituted by one or more substituents selected from the groupconsisting of halogen, hydroxyl, a C₁-C₈ alkoxy, amino, carboxy, sulfo,and phenyl, where the phenyl is optionally substituted by one or moresubstituents selected from the group consisting of halogen, hydroxyl, aC₁-C₈ alkoxy, amino, a C₁-C₈ aminoalkyl, a C₁-C₈ sulfoalkyl, and a C₁-C₈carboxyalkyl; or one or more R²¹, R²², and R²³, taken in combinationwith R²⁰ and SPACER, or with SPACER alone, forms a 5- or 6-memberedring.
 7. The method according to claim 6, wherein R⁴ or R⁵ isindependently a TAIL or a CYCLIC SUBSTITUENT substituted by TAIL.
 8. Themethod according to claim 7, wherein R⁴ is a TAIL and R⁵ is a C₁-C₆alkyl group.
 9. The method according to claim 8, wherein CAP is—NR²¹R²², where R²¹ and R²² are independently C₁-C₆ alkyl groups. 10.The method according to claim 8, wherein CAP is —NR²¹R²²R²³, where²¹R²², and R²³ are independently C₁-C₆ alkyl groups.
 11. The methodaccording to claim 7, wherein R⁵ is a CYCLIC SUBSTITUENT that is an arylor heteroaryl and R⁴ is a TAIL, where CAP is —OR²¹ or —SR²¹.
 12. Themethod according to claim 7, wherein R⁵ is a TAIL.
 13. The methodaccording to claim 12, wherein CAP is —NR²¹R²²R²³, where R²¹, R²² andR²³ are independently C₁-C₆ alkyl groups.
 14. The method according toclaim 1, wherein the staining solution comprises one or more dyes havingthe structure of Compound 15, Compound 16, Compound 22, Compound 23,Compound 24, or Compound
 25. 15. The method according to claim 1,wherein the double stranded DNA is in solution, immobilized on a solidor semi-solid matrix, or present in a biological structure.
 16. Themethod according to claim 1, further comprising quantifying thedouble-stranded DNA present in the sample.
 17. The method according toclaim 1, wherein the fluorescence of the dye when associated withdouble-stranded DNA is distinguishable from the fluorescence of the dyewhen associated with single-stranded nucleic acid.
 18. The methodaccording to claim 15, wherein the biological structure is a biologicalcell or portion thereof, virus particle or tissue section.
 19. Themethod according to claim 15, wherein the double-stranded DNA is insolution that is free from cells or portions thereof.
 20. The methodaccording to claim 15, wherein the solid or semi-solid matrix is apolymeric gel, an array, a glass slide, a polymeric microparticle, amembrane a silica-based support, or a plastic-based support.